G-CSF conjugates

ABSTRACT

Polypeptide conjugates with G-CSF activity comprising a polypeptide having at least one introduced lysine residue and at least one removed lysine residue compared to the sequence of human G-CSF, and which are conjugated to 2-6 polyethylene glycol moieties. The conjugates have a low in vitro bioactivity, a long in vivo half-life, a reduced receptor-mediated clearance, and provide a more rapid stimulation of production of white blood cells and neutrophils than non-conjugated recombinant human G-CSF.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of co-pending U.S.application Ser. No. 09/904,196 filed Jul. 11, 2001. Pursuant to 35U.S.C. §119(a)-(d), this application also claims priority from andbenefit of Danish Patent Application No. PA 2002 00447 filed Mar. 22,2002, and Danish Patent Application No. PA 2002 00708 filed May 8, 2002.U.S. Ser. No. 09/904,196 is a continuation-in-part of U.S. applicationSer. No. 09/760,008 filed Jan. 10, 2001. U.S. Ser. No. 09/760,008 claimspriority to and benefit of U.S. Provisional Application Serial No.60/176,376 filed Jan. 14, 2000, U.S. Provisional Application Serial No.60/189,506 filed Mar. 15, 2000, U.S. Provisional Application Serial No.60/215,644 filed Jun. 30, 2000, Danish Patent Application No. PA 200000024 filed Jan. 10, 2000, Danish Patent Application No. PA 2000 00341filed Mar. 2, 2000, and Danish Patent Application No. PA 2000 00943filed Jun. 16, 2000. The disclosure of each application listed above isincorporated herein in its entirety for all purposes.

COPYRIGHT NOTIFICATION

[0002] Pursuant to 37 C.F.R. §1.71(e), Applicants note that a portion ofthis disclosure contains material which is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosure,as it appears in the Patent and Trademark Office patent file or records,but otherwise reserves all copyright rights whatsoever.

FIELD OF THE INVENTION

[0003] The present invention relates to new polypeptides exhibitinggranulocyte colony-stimulating factor (G-CSF) activity, to conjugatesbetween a polypeptide exhibiting G-CSF activity and a non-polypeptidemoiety, to methods for preparing such polypeptides or conjugates and theuse of such polypeptides or conjugates in therapy, in particular for thetreatment of neutropenia or leukopenia.

BACKGROUND OF THE INVENTION

[0004] The process by which white blood cells grow, divide anddifferentiate in the bone marrow is called hematopoiesis (Dexter andSpooncer, Ann. Rev. Cell. Biol., 3:423, 1987). Each of the blood celltypes arises from pluripotent stem cells. There are generally threeclasses of blood cells produced in vivo: red blood cells (erythrocytes),platelets and white blood cells (leukocytes), the majority of the latterbeing involved in host immune defense. Proliferation and differentiationof hematopoietic precursor cells are regulated by a family of cytokines,including colony-stimulating factors (CSF's) such as G-CSF andinterleukins (Arai et al., Ann. Rev. Biochem., 59:783-836, 1990). Theprincipal biological effect of G-CSF in vivo is to stimulate the growthand development of certain white blood cells known as neutrophilicgranulocytes or neutrophils (Welte et al., PNAS-USA 82:1526-1530, 1985,Souza et al., Science, 232:61-65, 1986). When released into the bloodstream, neutrophilic granulocytes function to fight bacterial and otherinfection.

[0005] The amino acid sequence of human G-CSF (hG-CSF) was reported byNagata et al. Nature 319:415-418, 1986. hG-CSF is a monomeric proteinthat dimerizes the G-CSF receptor by formation of a 2:2 complex of 2G-CSF molecules and 2 receptors (Horan et al. (1996), Biochemistry35(15): 4886-96). Aritomi et al. Nature 401:713-717, 1999 have describedthe X-ray structure of a complex between hG-CSF and the BN-BC domains ofthe G-CSF receptor. They identify the following hG-CSF residues as beingpart of the receptor binding interfaces: G4, P5, A6, S7, S8, L9, P10,Q11, S12, L15, K16, E19, Q20, L108, D109, D112, T115, T116, Q119, E122,E123, and L124. Expression of rhG-CSF in Escherichia coli, Saccharomycescerevisiae and mammalian cells has been reported (Souza et al., Science232:61-65, 1986, Nagata et al., Nature 319: 415-418, 1986, Robinson andWittrup, Biotechnol. Prog. 11:171-177, 1985).

[0006] Leukopenia (a reduced level of white blood cells) and neutropenia(a reduced level of neutrophils) are disorders that result in anincreased susceptibility to various types of infections. Neutropenia canbe chronic, e.g. in patients infected with HIV, or acute, e.g. in cancerpatients undergoing chemotherapy or radiation therapy. For patients withsevere neutropenia, e.g. as a result of chemotherapy, even relativelyminor infections can be serious and even life-threatening. Recombinanthuman G-CSF (rhG-CSF) is generally used for treating various forms ofleukopenia/neutropenia. Thus, commercial preparations of rhG-CSF areavailable under the names filgrastim (Gran® and Neupogen®), lenograstim(Neutrogin® and Granocyte®) and nartograstim (Neu-up®). Gran® andNeupogen® are non-glycosylated and produced in recombinant E. colicells. Neutrogin® and Granocyte® are glycosylated and produced inrecombinant CHO cells and Neu-up® is non-glycosylated with five aminoacids substituted at the N-terminal region of intact rhG-CSF produced inrecombinant E. coli cells.

[0007] Various protein-engineered variants of hG-CSF have been reported(e.g. U.S. Pat. Nos. 5,581,476, U.S. 5,214,132, U.S. 5,362,853, U.S.4,904,584 and Riedhaar-Olson et al. Biochemistry 35: 9034-9041, 1996).Modification of hG-CSF and other polypeptides so as to introduce atleast one additional carbohydrate chain as compared to the nativepolypeptide has been suggested (U.S. Pat. No. 5,218,092). It is statedthat the amino acid sequence of the polypeptide may be modified by aminoacid substitution, amino acid deletion or amino acid insertion so as toeffect addition of an additional carbohydrate chain. In addition,polymer modifications of native hG-CSF, including attachment of PEGgroups, have been reported (Satake-Ishikawa et al., Cell Structure andFunction 17:157-160, 1992, U.S. Pat. No. 5,824,778, U.S. Pat. No.5,824,784, WO 96/11953, WO 95/21629, WO 94/20069).

[0008] Bowen et al., Experimental Hematology 27 (1999), 425-432 disclosea study of the relationship between molecule mass and duration ofactivity of PEG-conjugated G-CSF mutein. An apparent inverse correlationwas suggested between molecular weight of the PEG moieties conjugated tothe protein and in vitro activity, whereas in vivo activities increasedwith increasing molecular weight. It is speculated that a lower affinityof the conjugates act to increase the half-life, becausereceptor-mediated endocytosis is an important mechanism regulatinglevels of hematopoietic growth factors.

[0009] The commercially available rhG-CSF has a short-termpharmacological effect and must therefore be administered once a day forthe duration of the leukopenic state. A molecule with a longercirculation half-life would decrease the number of administrationsnecessary to alleviate the leukopenia and prevent consequent infections.Another, more significant problem with currently available rG-CSFproducts is that patients become neutropenic after chemotherapy evenafter administration of G-CSF. For these patients, it is important to beable to reduce the duration and degree of the neutropenic state as muchas possible in order to minimize the risk of serious infections. Afurther problem is the occurrence of dose-dependent bone pain. Sincebone pain is experienced by patients as a significant side effect oftreatment with rG-CSF, it would be desirable to provide a rG-CSF productthat does not cause bone pain, either by means of a product thatinherently does not have this effect or that is effective in asufficiently small dose that no bone pain is caused. Thus, there isclearly a need for improved recombinant G-CSF-like molecules.

[0010] With respect to the half-life, one way to increase thecirculation half-life of a protein is to ensure that clearance of theprotein, in particular via renal clearance and receptor-mediatedclearance, is reduced. This may be achieved by conjugating the proteinto a chemical moiety which is capable of increasing the apparent size,thereby reducing renal clearance and increasing the in vivo half-life.Furthermore, attachment of a chemical moiety to the protein mayeffectively block proteolytic enzymes from physical contact with theprotein, thus preventing degradation by non-specific proteolysis.Polyethylene glycol (PEG) is one such chemical moiety that has been usedin the preparation of therapeutic protein products. Recently, G-CSFmolecule modified with a single, N-terminally linked 20 kDa PEG group(Neulastam) was approved for sale in the United States. This PEGylatedG-CSF molecule has been shown to have an increased half-life compared tonon-PEGylated G-CSF and thus may be administered less frequently thancurrent G-CSF products, but it does not reduce the duration ofneutropenia significantly compared to non-PEGylated G-CSF. Thus, thereis still substantial room for improvement of the known G-CSF molecules.

[0011] A need therefore still exists for providing novel moleculesexhibiting G-CSF activity that are useful in the treatment ofleukopenia/neutropenia, and which have are improved in terms of e.g. anincreased half-life and in particular a reduction in the duration ofneutropenia. The present invention relates to such molecules.

BRIEF DISCLOSURE OF THE INVENTION

[0012] The present invention relates to specific conjugates comprising apolypeptide exhibiting G-CSF activity and a non-polypeptide moiety,methods for their preparation and their use in medical treatment and inthe preparation of pharmaceuticals. Accordingly, in a first aspect theinvention relates to various specific conjugates comprising apolypeptide exhibiting G-CSF activity and having an amino acid sequencethat differs from the known amino acid sequence of human G-CSF as shownin SEQ ID NO: 1 in at least one specified altered amino acid residuecomprising an attachment group for a non-polypeptide moiety, and havingat least one non-polypeptide moiety attached to an attachment group ofthe polypeptide. These conjugates have a substantially reduced in vitrobioactivity compared to that of non-conjugated hG-CSF, whichsurprisingly has been shown to result in a more rapid neutrophilrecovery. The conjugate of the present invention thus has one or moreimproved properties as compared to commercially available rhG-CSF,including increased stimulation of neutrophils, increased functional invivo half-life, increased serum half-life, reduced side effects, reducedimmunogenicity and/or increased bioavailability. Consequently, medicaltreatment with a conjugate of the invention offers a number ofadvantages over the currently available G-CSF compounds.

[0013] In a further aspect the invention relates to polypeptidesexhibiting G-CSF activity and which form part of a conjugate of theinvention. The polypeptides of the invention are contemplated to beuseful as such for therapeutic, diagnostic or other purposes, but findparticular interest as intermediate products for the preparation of aconjugate of the invention.

[0014] In a further aspect the invention relates to a polypeptideconjugate comprising a polypeptide exhibiting G-CSF activity, whichcomprises an amino acid sequence that differs from the amino acidsequence of hG-CSF (with the amino acid sequence shown in SEQ ID NO: 1)in at least one amino acid residue selected from an introduced orremoved amino acid residue comprising an attachment group for anon-polypeptide moiety, and a sufficient number or type ofnon-polypeptide moieties to provide the conjugate with an increasedhalf-life and/or a more rapid neutrophil recovery compared to knownrecombinant G-CSF products.

[0015] In a particular aspect the invention relates to a polypeptideconjugate exhibiting G-CSF activity, comprising a polypeptide having thesubstitutions K16R, K34R, K40R, T105K, and S159K, and optionally asubstitution in position H170, e.g. to R, K or Q, relative to the aminoacid sequence of hG-CSF shown in SEQ ID NO: 1, or in a correspondingposition relative to an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 1, and having 2-6, typically 3-6 polyethyleneglycol moieties with a molecular weight of about 1000-10,000 Da attachedto one or more attachment groups of the polypeptide. Where thesesubstitutions are relative to a sequence with at least about 80%sequence identity with SEQ ID NO: 1, the degree of sequence identity istypically at least about 90% or 95%, such as at least about 96%, 97% 98%or 99%.

[0016] In still further aspects the invention relates to methods forpreparing a conjugate of the invention, including nucleotide sequencesencoding a polypeptide of the invention, expression vectors comprisingsuch a nucleotide sequence, and host cells comprising such a nucleotidesequence or expression vector.

[0017] In final aspects the invention relates to a compositioncomprising a conjugate or polypeptide of the invention, a method forpreparing a pharmaceutical composition, use of a conjugate orcomposition of the invention as a pharmaceutical, and a method oftreating a mammal with such composition. In particular, the polypeptide,conjugate or composition of the invention may be used to preventinfection in cancer patients undergoing certain types of radiationtherapy, chemotherapy, and bone marrow transplantations, to mobilizeprogenitor cells for collection in peripheral blood progenitor celltransplantations, for treatment of severe chronic or relativeleukopenia, irrespective of cause, and to support treatment of patientswith acute myeloid leukemia. Additionally, the polypeptide, conjugate orcomposition of the invention may be used for treatment of AIDS or otherimmunodeficiency diseases as well as bacterial infections.

DETAILED DISCLOSURE OF THE INVENTION

[0018] Definitions

[0019] In the context of the present application and invention thefollowing definitions apply:

[0020] The term “conjugate” is intended to indicate a heterogeneousmolecule formed by the covalent attachment of one or more polypeptides,typically a single polypeptide, to one or more non-polypeptide moietiessuch as polymer molecules, lipophilic compounds, carbohydrate moietiesor organic derivatizing agents. The term covalent attachment means thatthe polypeptide and the non-polypeptide moiety are either directlycovalently joined to one another, or else are indirectly covalentlyjoined to one another through an intervening moiety or moieties, such asa bridge, spacer, or linkage moiety or moieties. Preferably, theconjugate is soluble at relevant concentrations and conditions, i.e.soluble in physiological fluids such as blood. The term “non-conjugatedpolypeptide” may be used about the polypeptide part of the conjugate.

[0021] The term “polypeptide” may be used interchangeably herein withthe term “protein”.

[0022] The “polymer molecule” is a molecule formed by covalent linkageof two or more monomers, wherein none of the monomers is an amino acidresidue, except where the polymer is human albumin or another abundantplasma protein. The term “polymer” may be used interchangeably with theterm “polymer molecule”. The term is intended to cover carbohydratemolecules, although, normally, the term is not intended to cover thetype of carbohydrate molecule which is attached to the polypeptide by invivo N— or O-glycosylation (as further described below), since suchmolecule is referred to herein as “an oligosaccharide moiety”. Exceptwhere the number of polymer molecule(s) is expressly indicated everyreference to “a polymer”, “a polymer molecule”, “the polymer” or “thepolymer molecule” contained in a polypeptide of the invention orotherwise used in the present invention shall be a reference to one ormore polymer molecule(s).

[0023] The term “attachment group” is intended to indicate an amino acidresidue group of the polypeptide capable of coupling to the relevantnon-polypeptide moiety. For instance, for polymer conjugation, inparticular to PEG, a frequently used attachment group is the ε-aminogroup of lysine or the N-terminal amino group. Other polymer attachmentgroups include a free carboxylic acid group (e.g. that of the C-terminalamino acid residue or of an aspartic acid or glutamic acid residue),suitably activated carbonyl groups, oxidized carbohydrate moieties andmercapto groups. Useful attachment groups and their matching non-peptidemoieties are apparent from the table below. Conjugation AttachmentExamples of non- method/- group Amino acid peptide moiety Activated PEGReference —NH₂ N-terminal, Polymer, e.g. PEG, mPEG-SPA Shearwater Corp.Lys, His, Arg with amide or imine Tresylated Delgado et al., criticalgroup mPEG reviews in Therapeutic Drug Carrier Systems9(3,4):249-304(1992) —COOH C-term, Asp, Polymer, e.g. PEG, mPEG-HzShearwater Corp. Glu with ester or amide In vitro coupling groupOligosaccharide moiety —SH Cys Polymer, e.g. PEG, PEG- Shearwater Corp.with disulfide, vinylsulphone Delgado et al., critical maleimide orvinyl PEG-maleimide reviews in Therapeutic sulfone group In vitrocoupling Drug Carrier Systems Oligosaccharide 9(3,4):249-304 (1992)moiety —OH Ser, Thr, -OH, Oligosaccharide In vivo O-linked Lys moietyglycosylation PEG with ester, ether, carbamate, carbonate —CONH₂ Asn aspart of Oligosaccharide In vivo N- an N- moiety glycosylationglycosylation Polymer, e.g. PEG site Aromatic Phe, Tyr, TrpOligosaccharide In vitro coupling residue moiety —CONH₂ GlnOligosaccharide In vitro coupling Yan and Wold, moiety Biochemistry,1984, Jul. 31; 23(16): 3759- 65 Aldehyde Oxidized Polymer, e.g. PEG,PEGylation Andresz et al., 1978, Ketone oligo- PEG-hydrazide Makromol.Chem. saccharide 179:301, WO 92/16555, WO 00/23114 Guanidino ArgOligosaccharide In vitro coupling Lundblad and Noyes, moiety ChemicalReagents for Protein Modification, CRC Press Inc., Florida, U.S.A.Imidazole His Oligosaccharide In vitro coupling As for guanidine ringmoiety

[0024] For in vivo N-glycosylation, the term “attachment group” is usedin an unconventional way to indicate the amino acid residuesconstituting an N-glycosylation site (with the sequence N-X′-S/T/C-X″,wherein X′ is any amino acid residue except proline, X″ any amino acidresidue which may or may not be identical to X′ and which preferably isdifferent from proline, N is asparagine, and S/T/C is either serine,threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is where the oligosaccharide moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site are present.Accordingly, when the non-peptide moiety is an oligosaccharide moietyand the conjugation is to be achieved by N-glycosylation, the term“amino acid residue comprising an attachment group for the non-peptidemoiety” as used in connection with alterations of the amino acidsequence of the polypeptide of interest is to be understood as meaningthat one or more amino acid residues constituting an N-glycosylationsite are to be altered in such a manner that either a functionalN-glycosylation site is introduced into the amino acid sequence orremoved from said sequence.

[0025] In the present application, amino acid names and atom names (e.g.CA, CB, NZ, N, O, C, etc.) are used as defined by the Protein DataBank(PDB) (www.pdb.org), which is based on the IUPAC nomenclature (IUPACNomenclature and Symbolism for Amino Acids and Peptides (residue names,atom names etc.), Eur. J. Biochem., 138, 9-37 (1984) together with theircorrections in Eur. J. Biochem., 152, 1 (1985). The term “amino acidresidue” is intended to indicate any naturally or non-naturallyoccurring amino acid residue, in particular an amino acid residuecontained in the group consisting of the 20 naturally occurring aminoacids, i.e. alanine (Ala or A), cysteine (Cys or C), aspartic acid (Aspor D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Glyor G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K),leucine (Leu or L), methionine (Met or M), asparagine (Asn or N),proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine(Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp orW), and tyrosine (Tyr or Y) residues.

[0026] The terminology used for identifying amino acidpositions/substitutions is illustrated as follows: F13 indicatesposition number 13 occupied by a phenylalanine residue in the referenceamino acid sequence. F13K indicates that the phenylalanine residue ofposition 13 has been substituted with a lysine residue. Unless otherwiseindicated, the numbering of amino acid residues made herein is maderelative to the amino acid sequence of hG-CSF shown in SEQ ID NO:1.Alternative substitutions are indicated with a “/”, e.g. Q67D/E means anamino acid sequence in which glutamine in position 67 is substitutedwith either aspartic acid or glutamic acid. Multiple substitutions areindicated with a “+”, e.g. S53N+G55S/T means an amino acid sequencewhich comprises a substitution of the serine residue in position 53 withan asparagine residue and a substitution of the glycine residue inposition 55 with a serine or a threonine residue.

[0027] The term “nucleotide sequence” is intended to indicate aconsecutive stretch of two or more nucleotide molecules. The nucleotidesequence may be of genomic, cDNA, RNA, semisynthetic or syntheticorigin, or any combination thereof.

[0028] The term “polymerase chain reaction” or “PCR” refers to thewell-known method for amplification of a desired nucleotide sequence invitro using a thermostable DNA polymerase.

[0029] “Cell”, “host cell”, “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing DNA into a cell.

[0030] “Operably linked” refers to the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. For example, the nucleotide sequenceencoding a presequence or secretory leader is operably linked to anucleotide sequence for a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide: a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the nucleotidesequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used, inconjunction with standard recombinant DNA methods.

[0031] The term “introduce” refers to introduction of an amino acidresidue comprising an attachment group for a non-polypeptide moiety, inparticular by substitution of an existing amino acid residue, oralternatively by insertion of an additional amino acid residue. The term“remove” refers to removal of an amino acid residue comprising anattachment group for a non-polypeptide moiety, in particular bysubstitution of the amino acid residue to be removed by another aminoacid residue, or alternatively by deletion (without substitution) of theamino acid residue to be removed.

[0032] When substitutions are performed in relation to a parentpolypeptide, they are preferably “conservative substitutions”, in otherwords substitutions performed within groups of amino acids with similarcharacteristics, e.g. small amino acids, acidic amino acids, polar aminoacids, basic amino acids, hydrophobic amino acids and aromatic aminoacids.

[0033] Preferred substitutions in the present invention may inparticular be chosen from among the conservative substitution groupslisted in the table below.

[0034] Conservative substitution groups: 1 Alanine (A) Glycine Serine(S) Threonine (G) (T) 2 Aspartic acid Glutamic (D) acid (E) 3 AsparagineGlutamine (N) (Q) 4 Arginine (R) Histidine Lysine (K) (H) 5 IsoleucineLeucine Methionine Valine (V) (I) (L) (M) 6 Phenylalanine TyrosineTryptophan (F) (Y) (W)

[0035] The term “immunogenicity” as used in connection with a givensubstance is intended to indicate the ability of the substance to inducea response from the immune system. The immune response may be a cell orantibody mediated response (see, e.g., Roitt: Essential Immunology(8^(th) Edition, Blackwell) for further definition of immunogenicity).Normally, reduced antibody reactivity will be an indication of reducedimmunogenicity. The reduced immunogenicity may be determined by use ofany suitable method known in the art, e.g. in vivo in vitro.

[0036] The term “functional in vivo half-life” is used in its normalmeaning, i.e. the time at which 50% of the biological activity of thepolypeptide or conjugate is still present in the body/target organ, orthe time at which the activity of the polypeptide or conjugate is 50% ofthe initial value. As an alternative to determining functional in vivohalf-life, “serum half-life” may be determined, i.e. the time in which50% of the polypeptide or conjugate molecules circulate in the plasma orbloodstream prior to being cleared. Alternative terms to serum half-lifeinclude “plasma half-life”, “circulating half-life”, “serum clearance”,“plasma clearance” and “clearance half-life”. The polypeptide orconjugate is cleared by the action of one or more of thereticuloendothelial systems (RES), kidney, spleen or liver, byreceptor-mediated degradation, or by specific or non-specificproteolysis, in particular by the action of receptor-mediated clearanceand renal clearance. Normally, clearance depends on size (relative tothe cutoff for glomerular filtration), charge, attached carbohydratechains, and the presence of cellular receptors for the protein. Thefunctionality to be retained is normally selected from proliferative orreceptor-binding activity. The functional in vivo half-life and theserum half-life may be determined by any suitable method known in theart as further discussed in the Materials and Methods section below.

[0037] The term “increased” as used about the functional in vivohalf-life or serum half-life is used to indicate that the relevanthalf-life of the conjugate or polypeptide is statistically significantlyincreased relative to that of a reference molecule, such as anon-conjugated hG-CSF (e.g. Neupogen®) as determined under comparableconditions. For instance, the relevant half-life may increased by atleast about 25%, such as by at least about 50%, e.g. by at least about100%, 200%, 500% or 1000%.

[0038] The term “renal clearance” is used in its normal meaning toindicate any clearance taking place by the kidneys, e.g. by glomerularfiltration, tubular excretion or tubular elimination. Renal clearancedepends on physical characteristics of the conjugate, including size(diameter), symmetry, shape/rigidity and charge. Reduced renal clearancemay be established by any suitable assay, e.g. an established in vivoassay. Typically, renal clearance is determined by administering alabeled (e.g. radioactive or fluorescent labeled) polypeptide conjugateto a patient and measuring the label activity in urine collected fromthe patient. Reduced renal clearance is determined relative to acorresponding reference polypeptide, e.g. the correspondingnon-conjugated polypeptide, a non-conjugated corresponding wild-typepolypeptide or another conjugated polypeptide (such as a conjugatedpolypeptide not according to the invention), under comparableconditions. Preferably, the renal clearance rate of the conjugate isreduced by at least 50%, preferably by at least 75%, and most preferablyby at least 90% compared to a relevant reference polypeptide.

[0039] Generally, activation of the receptor is coupled toreceptor-mediated clearance (RMC) such that binding of a polypeptide toits receptor without activation does not lead to RMC, while activationof the receptor leads to RMC. The clearance is due to internalization ofthe receptor-bound polypeptide with subsequent lysosomal degradation.Reduced RMC may be achieved by designing the conjugate so as to be ableto bind and activate a sufficient number of receptors to obtain optimalin vivo biological response and avoid activation of more receptors thanrequired for obtaining such response. This may be reflected in reducedin vitro bioactivity and/or increased off-rate. In a preferredembodiment, the conjugates of the invention have a substantially reducedin vitro bioactivity compared to that of non-conjugated hG-CSF.

[0040] Typically, reduced in vitro bioactivity reflects reducedefficacy/efficiency and/or reduced potency and may be determined by anysuitable method for determining any of these properties. For instance,in vitro bioactivity may be determined in a luciferase based assay(“Primary assay 2”; see Materials and Methods). Another method fordetermining the in vitro bioactivity is to determine the bindingaffinity of a conjugate of the invention using the cell-based assaydescribed in the Materials and Methods section (“Secondary assay”).

[0041] It has been found that a relatively low in vitro bioactivity,compared to the activity of hG-CSF (SEQ ID NO:1), is advantageous interms of both a long plasma half-life and a high degree of stimulationof neutrophils. Surprisingly, it has been found that administration ofG-CSF conjugates of the invention having a low in vitro bioactivityresults in a faster neutrophil recovery, i.e. a faster recovery of theneutrophil count to a normal level, than administration of hG-CSF. Sinceit is critical to be able to reduce the duration of neutropenia as muchas possible in patients having a reduced neutrophil level due to e.g.chemotherapy or radiation therapy, this is an important finding. Thus,in a preferred embodiment, the in vitro bioactivity of a conjugate ofthe invention is in the range of about 2-30%, preferably about 3-25%, ofthe bioactivity of hG-CSF (where the hG-CSF used as the referencepolypeptide has SEQ ID NO:1, optionally with an N-terminal methionineresidue; the reference hG-CSF may in particular be Neupogen®, i.e.non-glycosylated Met-hG-CSF) as determined by the luciferase assaydescribed herein, or, alternatively, using the cell-based receptorbinding affinity assay (“Secondary assay”). The in vitro bioactivity ofthe conjugate is thus preferably reduced by at least 70%, such as by atleast 75%, e.g. by at least 80% or 85%, as compared to the in vitrobioactivity of hG-CSF, determined under comparable conditions. Expresseddifferently, the conjugate may have an in vitro bioactivity that is assmall as about 2%, typically at least about 3%, such as at least about4% or 5%, of that of the wild-type polypeptide. For instance, the invitro bioactivity may be in the range of about 4-20% of that of hG-CSF,determined under comparable conditions. In cases where reduced in vitrobioactivity is desired in order to reduce receptor-mediated clearance,it will be clear that sufficient bioactivity to obtain the desiredreceptor activation must nevertheless be maintained, which is why thebioactivity should be at least about 2% of that of hG-CSF and preferablyslightly higher as explained above.

[0042] It has been found that amino acid alterations, in particularsubstitutions, in the helix regions of G-CSF, i.e. in an amino acidresidue selected from amino acid position 11-41 (helix A), 71-95 (helixB), 102-125 (helix C), and 145-170 (helix D) (compared to SEQ ID NO:1),result in a reduced receptor-mediated clearance and thus an increased invivo half-life when the resulting polypeptides are conjugated topolyethylene glycol. In addition to a longer half-life, it hassurprisingly been found that administration of such polypeptideconjugates is able to stimulate production of white blood cells andneutrophils to the same degree as, or even better than, administrationof the commercially available G-CSF products Neupogen® and Neulasta™.G-CSF conjugates having a reduced in vitro bioactivity may thus beprepared by altering, typically by substitution, one or more amino acidresidues in a helix region of G-CSF, and by conjugating the resultingpolypeptide to one or more non-polypeptide moieties such as polyethyleneglycol.

[0043] Preferably, the off-rate between the polypeptide conjugate andits receptor is increased by a magnitude resulting in the polypeptideconjugate being released from its receptor before any substantialinternalization of the receptor-ligand complex has taken place. Thereceptor-polypeptide binding affinity may be determined as described inthe Materials and Methods section herein. The off-rate may be determinedusing the Biacore® technology as described in the Materials and Methodssection. The in vitro RMC may be determined by labeling (e.g.radioactive or fluorescent labeling) the polypeptide conjugate,stimulating cells comprising the receptor for the polypeptide, washingthe cells, and measuring label activity. Alternatively, the conjugatemay be exposed to cells expressing the relevant receptor. After anappropriate incubation time the supernatant is removed and transferredto a well containing similar cells. The biological response of thesecells to the supernatant is determined relative to a non-conjugatedpolypeptide or another reference polypeptide, and this is a measure ofthe extent of the reduced RMC.

[0044] Normally, reduced in vitro bioactivity of the conjugate isobtained as a consequence of its modification by a non-polypeptidemoiety. However, in order to further reduce in vitro bioactivity or forother reasons it may be of interest to modify the polypeptide part ofthe conjugate further. For instance, in one embodiment at least oneamino acid residue located at or near a receptor binding site of thepolypeptide may be substituted with another amino acid residue ascompared to the corresponding wild-type polypeptide so as to obtainreduced in vitro bioactivity. The amino acid residue to be introduced bysubstitution may be any amino acid residue capable of reducing in vitrobioactivity of the conjugate. Conveniently, the introduced amino acidresidue comprises an attachment group for the non-polypeptide moiety asdefined herein. In particular, when the non-polypeptide moiety is apolymer molecule such as PEG molecule, the amino acid residue to beintroduced may be a lysine residue.

[0045] The term “exhibiting G-CSF activity” is intended to indicate thatthe polypeptide or conjugate has one or more of the functions of nativeG-CSF, in particular hG-CSF with the amino acid sequence shown in SEQ IDNO: 1, including the capability to bind to a G-CSF receptor (Fukunaga etal., J. Bio. Chem, 265:14008, 1990). The G-CSF activity is convenientlyassayed using the primary assay described in the Materials and Methodssection hereinafter. The polypeptide “exhibiting” G-CSF activity isconsidered to have such activity when it displays a measurable function,e.g. a measurable proliferative activity or a receptor binding activity(e.g. as determined by the primary assay described in the Materials andMethods section). The polypeptide exhibiting G-CSF activity may also betermed “G-CSF molecule” herein for the sake of simplicity, even thoughsuch polypeptides are in fact variants of G-CSF.

[0046] The term “parent G-CSF” or “parent polypeptide” is intended toindicate the molecule to be modified in accordance with the presentinvention. The parent G-CSF is normally hG-CSF or a variant thereof. A“variant” is a polypeptide which differs in one or more amino acidresidues from a parent polypeptide, normally in 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15 amino acid residues. Examples of rhG-CSFinclude filgrastim (Gran® and Neupogen®), lenograstim (Neutrogin® andGranocyte®) and nartograstim (Neu-up®).

[0047] Conjugate of the Invention

[0048] As stated above, in a first aspect the invention relates to aconjugate comprising a polypeptide exhibiting G-CSF activity, whichcomprises an amino acid sequence that differs from the amino acidsequence of SEQ ID NO: 1 in at least one amino acid residue selectedfrom specified introduced or removed amino acid residues comprising anattachment group for a non-polypeptide moiety, and at least onenon-polypeptide moiety attached to an attachment group of thepolypeptide. The amino acid residues to be introduced and/or removed aredescribed in further detail in the following sections. It will beunderstood that the conjugate itself also exhibits G-CSF activity.

[0049] By removing and/or introducing an amino acid residue comprisingan attachment group for the non-polypeptide moiety it is possible tospecifically adapt the polypeptide so as to make the molecule moresusceptible to conjugation to the non-polypeptide moiety of choice, tooptimize the conjugation pattern (e.g. to ensure an optimal distributionof non-polypeptide moieties on the surface of the G-CSF molecule and toensure that only the attachment groups intended to be conjugated arepresent in the molecule) and thereby obtain a new conjugate moleculewhich has G-CSF activity and in addition one or more improved propertiesas compared to G-CSF molecules available today.

[0050] While the polypeptide may be of any origin, in particularmammalian origin, it is presently preferred to be of human origin, inparticular a variant of a polypeptide having the amino acid sequence ofSEQ ID NO: 1.

[0051] In preferred embodiments of the present invention more than oneamino acid residue of the polypeptide with G-CSF activity is altered,e.g. the alteration embraces removal as well as introduction of aminoacid residues comprising an attachment group for the non-polypeptidemoiety of choice.

[0052] In addition to the amino acid alterations disclosed herein aimedat removing and/or introducing attachment sites for the non-polypeptidemoiety, it will be understood that the amino acid sequence of thepolypeptide of the invention may if desired contain other alterationsthat need not be related to introduction or removal of attachment sites,i.e. other substitutions, insertions or deletions. These may, forexample, include truncation of the N- and/or C-terminus by one or moreamino acid residues, or addition of one or more extra residues at the N-and/or C-terminus, e.g. addition of a methionine residue at theN-terminus.

[0053] The conjugate of the invention has one or more of the followingimproved properties as compared to hG-CSF, in particular as compared torhG-CSF (e.g. filgrastim, lenograstim or nartograstim) or known hG-CSFvariants: increased ability to reduce the duration of neutropenia,increased functional in vivo half-life, increased serum half-life,reduced renal clearance, reduced receptor-mediated clearance, reducedside effects such as bone pain, and reduced immunogenicity.

[0054] It will be understood that the amino acid residue comprising anattachment group for a non-polypeptide moiety, whether it be removed orintroduced, will be selected on the basis of the nature of thenon-polypeptide moiety of choice and, in most instances, on the basis ofthe method by which conjugation between the polypeptide and thenon-polypeptide moiety is to be achieved. For instance, when thenon-polypeptide moiety is a polymer molecule such as a polyethyleneglycol or polyalkylene oxide derived molecule amino acid residuescomprising an attachment group may be selected from the group consistingof lysine, cysteine, aspartic acid, glutamic acid, histidine andarginine. When conjugation to a lysine residue is to be achieved, asuitable activated molecule is e.g. mPEG-SPA from Shearwater Corp.,oxycarbonyl-oxy-N-dicarboxyimide-PEG (U.S. Pat. No. 5,122,614), or PEGavailable from PolyMASC Pharmaceuticals plc. The first of these will beillustrated further below.

[0055] In order to avoid too much disruption of the structure andfunction of the parent hG-CSF molecule, the total number of amino acidresidues to be altered in accordance with the present invention, e.g. asdescribed in the subsequent sections herein, (as compared to the aminoacid sequence shown in SEQ ID NO:1) will typically not exceed 15. Theexact number of amino acid residues and the type of amino acid residuesto be introduced or removed depends in particular on the desired natureand degree of conjugation (e.g. the identity of the non-polypeptidemoiety, how many non-polypeptide moieties it is desirable or possible toconjugate to the polypeptide, where conjugation is desired or should beavoided, etc.). Preferably, the polypeptide part of the conjugate of theinvention or the polypeptide of the invention comprises an amino acidsequence which differs in 1-15 amino acid residues from the amino acidsequence shown in SEQ ID NO:1, typically in 2-10 amino acid residues,e.g. in 3-8 amino acid residues, such as 4-6 amino acid residues, fromthe amino acid sequence shown in SEQ ID NO: 1. Thus, normally thepolypeptide part of the conjugate or the polypeptide of the inventioncomprises an amino acid sequence which differs from the amino acidsequence shown in SEQ ID NO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 amino acid residues.

[0056] The polypeptide part of the conjugate will typically have anamino acid sequence with at least about 80% identity with SEQ ID NO: 1,preferably at least about 90%, such as at least about 95%, e.g. at leastabout 96%, 97%, 98% or 99% sequence identity with SEQ ID is NO: 1. Aminoacid sequence homology/identity is conveniently determined from alignedsequences, using e.g. the ClustalW program, version 1.8, June 1999,using default parameters (Thompson et al., 1994, ClustalW: Improving thesensitivity of progressive multiple sequence alignment through sequenceweighting, position-specific gap penalties and weight matrix choice,Nucleic Acids Research, 22: 4673-4680) or from the PFAM familiesdatabase version 4.0 (http://pfam.wustl.edu/) (Nucleic Acids Res. 1999Jan 1; 27(1):260-2) by use of GENEDOC version 2.5 (Nicholas, K. B.,Nicholas H. B. Jr., and Deerfield, D. W. II. 1997 GeneDoc: Analysis andVisualization of Genetic Variation, EMBNEW.NEWS 4:14; Nicholas, K. B.and Nicholas H. B. Jr. 1997 GeneDoc: Analysis and Visualization ofGenetic Variation).

[0057] In a preferred embodiment one difference between the amino acidsequence of the polypeptide and the amino acid sequence shown in SEQ IDNO: 1 is that at least one and often more, e.g. 1-15, amino acidresidues comprising an attachment group for the non-polypeptide moietyhas been introduced, preferably by substitution, into the amino acidsequence. Thereby, the polypeptide part is altered in the content of thespecific amino acid residues to which the non-polypeptide moiety ofchoice binds, whereby a more efficient, specific and/or extensiveconjugation is achieved. For instance, when the total number of aminoacid residues comprising an attachment group for the non-polypeptide ofchoice is altered to an optimized level, the clearance of the conjugateis typically significantly reduced, due to the altered shape, sizeand/or charge of the molecule achieved by the conjugation. Furthermore,when the total number of amino acid residues comprising an attachmentgroup for the non-polypeptide of choice is increased, a greaterproportion of the polypeptide molecule is shielded by thenon-polypeptide moieties of choice, leading to a lower immune response.

[0058] The term “one difference” as used in the present application isintended to allow for additional differences being present. Accordingly,in addition to the specified amino acid difference, other amino acidresidues than those specified may be mutated.

[0059] In a further preferred embodiment one difference between theamino acid sequence of the polypeptide and the amino acid sequence shownin SEQ ID NO: 1 is that at least one and preferably more, e.g. 1-15,amino acid residues comprising an attachment group for thenon-polypeptide moiety has/have been removed, preferably bysubstitution, from the amino acid sequence. By removing one or moreamino acid residues comprising an attachment group for thenon-polypeptide moiety of choice it is possible to avoid conjugation tothe non-polypeptide moiety in parts of the polypeptide in which suchconjugation is disadvantageous, e.g. in amino acid residues located ator near a functional site of the polypeptide (since conjugation at sucha site may result in inactivation or reduced G-CSF activity of theresulting conjugate due to impaired receptor recognition). In thepresent context the term “functional site” is intended to indicate oneor more amino acid residues which is/are essential for or otherwiseinvolved in the function or performance of hG-CSF. Such amino acidresidues are a part of the functional site. The functional site may bedetermined by methods known in the art and is preferably identified byanalysis of a structure of the polypeptide complexed to a relevantreceptor, such as the hG-CSF receptor (See Aritomi et al. Nature401:713-717, 1999).

[0060] In a still further preferred embodiment, the amino acid sequenceof the polypeptide differs from the amino acid sequence shown in SEQ IDNO: 1 in that a) at least one specified amino acid residue comprising anattachment group for the non-polypeptide moiety and present in the aminoacid sequence shown in SEQ ID NO: 1 has been removed, preferably bysubstitution, and b) at least one specified amino acid residuecomprising an attachment group for the non-polypeptide moiety has beenintroduced into the amino acid sequence, preferably by substitution, thespecified amino acid residues being any of those described in thesubsequent sections herein. This embodiment is considered of particularinterest in that it is possible to specifically design the polypeptideso as to obtain an optimal conjugation to the non-polypeptide moiety ofchoice. For instance, by introducing and removing selected amino acidresidues as disclosed in the following sections it is possible to ensurean optimal distribution of attachment groups for the non-polypeptidemoiety of choice, which gives rise to a conjugate in which thenon-polypeptide moieties are placed so as to a) effectively shieldepitopes and other surface parts of the polypeptide and b) ensure anoptimal Stokes radius of the conjugate, without causing too muchstructural disruption and thereby impair the function of thepolypeptide.

[0061] The conjugate of the invention will in general comprise asufficient number and type of non-polypeptide moieties to provide theconjugate with an increased functional in vivo half-life and/or serumhalf-life as compared to hG-CSF, e.g. filgrastim, lenograstim ornartograstim, and preferably as compared to rhG-CSF comprising a singleN-terminally attached 20 kDa PEG moiety. The increased functional invivo half-life is conveniently determined as described in the Materialsand Methods section herein.

[0062] The conjugate of the invention may comprise at least onenon-conjugated, conjugatable attachment group for the non-polypeptidemoiety. In the present context the term “conjugatable attachment group”is intended to indicate an attachment group that is located in aposition of the polypeptide where it is accessible for conjugation, andthat but for special precautions is conjugated to the relevantnon-polypeptide moiety when subjected to conjugation. For instance, suchattachment group may be part of an amino acid residue involved in orotherwise essential for the polypeptide to exert its activity. Aconvenient way to avoid conjugation of an otherwise conjugatableattachment group is to shield the attachment group by means of a helpermolecule, e.g. as described in the section entitled “Blocking of thefunctional site”. It will be understood that the number ofnon-conjugated, conjugatable attachment groups depends on the specificG-SCF polypeptide and the location of the conjugatable attachmentgroups. For instance, the polypeptide conjugate comprises one or twonon-conjugated, conjugatable attachment groups, and at least one, andpreferably two or more conjugated attachment groups.

[0063] The four helices of G-CSF comprise amino acid residues 11-41(helix A), 71-95 (helix B), 102-125 (helix C), and 145-170 (helix D)(Zink et al. (1994) Biochemistry 33: 8453-8463). Surprisingly, it hasbeen found that advantageous results may be obtained whennon-polypeptide moieties are attached to amino acid residues located inone or more of the helices of G-CSF, even though modification of proteinhelices, e.g. the helix structures of four-helix bundle proteins such asG-CSF, is generally considered to be accompanied by a risk ofdisturbance of protein function. In one embodiment, the polypeptideconjugate of the invention therefore comprises at least onenon-polypeptide moiety attached to an attachment group of an amino acidresidue located in one of the four helices, in particular in one or moreof the B, C or D helices

[0064] Conjugate of the Invention, Wherein the Non-Polypeptide Moiety isAttached to a Lysine or the N-terminal Amino Acid Residue

[0065] In one aspect the invention relates to a polypeptide conjugatecomprising

[0066] i) a polypeptide exhibiting G-CSF activity, comprising an aminoacid sequence that differs from the amino acid sequence shown in SEQ IDNO: 1 in at least one substitution selected from the group consisting ofT1K, P2K, L3K, G4K, P5K, A6K, S7K, S8K, L9K, P10K, Q11K, S12K, F13K,L14K, L15K, E19K, Q20K, V21K, Q25K, G26K, D27K, A29K, A30K, E33K, A37K,T38K, Y39K, L41K, H43K, P44K, E45K, E46K, V48K, L49K, L50K, H52K, S53K,L54K, 156K, P57K, P60K, L61K, S62K, S63K, P65K, S66K, Q67K, A68K, L69K,Q70K, L71K, A72K, G73K, S76K, Q77K, L78K, S80K, F83K, Q86K, G87K, Q90K,E93K, G94K, S96K, P97K, E98K, L99K, G100K, P101K, T102K, D104K, T105K,Q107K, L108K, D109K, A111K, D112K, F113K, T115K, T116K, W118K, Q119K,Q120K, M121K, E122K, E123K, L124K, M126K, A127K, P128K, A129K, L130K,Q131K, P132K, T133K, Q134K, G135K, A136K, M137K, P138K, A139K, A141K,S142K, A143K, F144K, Q145K, S155K, H156K, Q158K, S159K, L161K, E162K,V163K, S164K, Y165K, V167K, L168K, H170K, L171K, A172K, Q173K and P174K,and

[0067] ii) at least one non-polypeptide moiety attached to a lysineresidue of the polypeptide.

[0068] hG-CSF contains four lysine residues, of which K16 is located inthe receptor-binding domain and the others are located in positions 23,34 and 40, respectively, all relatively close to the receptor-bindingdomain. In order to avoid conjugation to one or more of these lysineresidues (since this may inactivate or severely reduce the activity ofthe resulting conjugate) it may be desirable to remove at least onelysine residue, e.g. two, three or all of these residues. Accordingly,in another, more preferred aspect the invention relates to a polypeptideconjugate as defined above, wherein at least one of the amino acidresidues selected from the group consisting of K16, K23, K34 and K40 hasbeen deleted or substituted with another amino acid residue. Preferably,at least K16 is substituted with another amino acid residue.

[0069] Examples of preferred amino acid substitutions include one ormore of Q70K, Q90K, T105K, Q120K, T133K, S159K and H170K/Q/R, such astwo, three, four or five of these substitutions, for example: Q70K+Q90K,Q70K+T105K, Q70K+Q120K, Q70K+T133K, Q70K+S159K, Q70K+H170K, Q90K+T105K,Q90K+Q120K, Q90K+T133K, Q90K+S159K, Q90K+H170K, T105K+Q120K,T105K+T133K, T105K+S159K, T105K+H170K, Q120K+T133K, Q120K+S159K,Q120K+H170K, T133K+S159K, T133K+H170K, S159K+H170K, Q70K+Q90K+T105K,Q70K+Q90K+Q120K, Q70K+Q90K+T133K, Q70K+Q90K+S159K, Q70K+Q90K+H170K,Q70K+T105K+Q120K, Q70K+T105K+T133K, Q70K+T105K+S159K, Q70K+T105K+H170K,Q70K+Q120K+T133K, Q70K+Q120K+S159K, Q70K+Q120K+H170K, Q70K+T133K+S159K,Q70K+T133K+H170K, Q70K+S159K+H170K, Q90K+T105K+Q120K, Q90K+T105K+T133K,Q90K+T105K+S159K, Q90K+T105K+H170K, Q90K+Q120K+T133K, Q90K+Q120K+S159K,Q90K+Q120K+H170K, Q90K+T133K+S159K, Q90K+T133K+H170K, Q90+S159K+H170K,T105K+Q120K+T133K, T105K+Q120K+S159K, T105K+Q120K+H170K,T105K+T133K+S159K, T105K+T133K+H170K, T105K+S159K+H170K,Q120K+T133K+S159K, Q120K+T133K+H170K, Q120K+S159K+H170K,T133K+S159K+H170K, Q70K+Q90K+T105K+Q120K, Q70K+Q90K+T105K+T133K,Q70K+Q90K+T105K+S159K, Q70K+Q90K+T105K+H170K, Q70K+Q90K+Q120K+T133K,Q70K+Q90K+Q120K+S159K, Q70K+Q90K+Q120K+H170K, Q70K+Q90K+T133K+S159K,Q70K+Q90K+T133K+H170K, Q70K+Q90K+S159K+H170K, Q70K+T105K+Q120K+T133K,Q70K+T105K+Q120K+S159K, Q70K+T105K+Q120K+H170K, Q70K+T105K+T133K+S159K,Q70K+T105K+T133K+H170K, Q70K+T105K+S159K+H170K, Q70K+Q120K+T133K+S159K,Q70K+Q120K+T133K+H170K, Q70K+T133K+S159K+H170K, Q90K+T105K+Q120K+T133K,Q90K+T105K+Q120K+S159K, Q90K+T105K+Q120K+H170K, Q90K+T105+T133K+S159K,Q90K+T105+T133K+H170K, Q90K+T105+S159K+H170K, Q90K+Q120K+T133K+S159K,Q90K+Q120K+T133K+H170K, Q90K+Q120K+S159K+H170K, Q90K+T133K+S159K+H170K,T105K+Q120K+T133K+S159K, T105K+Q120K+T133K+H170K,T105K+Q120K+S159K+H170K, T105K+T133K+S159K+H170K orQ120K+T133K+S159K+H170K. In any of the variants listed above, thesubstitution H170K may instead be HI 70Q or H170R.

[0070] The polypeptide of the conjugate according to this aspect of theinvention, i.e. having at least one introduced and one removed lysine,preferably comprises at least one, such as one, two, three or four, ofthe substitutions selected from the group consisting of K16R, K16Q,K23R, K23Q, K34R, K34Q, K40R and K40Q, preferably at least thesubstitution K16R, whereby conjugation of this residue can be avoided.Preferably, the polypeptide comprises at least one substitution selectedfrom the group consisting of K16R+K23R, K16R+K34R, K16R+K40R, K23R+K34R,K23R+K40R, K34R+K40R, K16R+K23R+K34R, K16R+K23R+K40R, K23R+K34R+K40R,K16R+K34R+K40R and K16R+K23R+K34R+K40R. In one preferred embodiment, thepolypeptide includes the substitutions K16R+K34R+K40R, while the lysinein position 23 is left unaltered. As indicated above, it is contemplatedthat any of the individual substitutions or combinations listed in thisparagraph for removal of a lysine residue may suitably be used with anyof the other substitutions disclosed herein for introduction of lysineresidues, in particular the substitutions listed in the paragraph above.

[0071] In a particular embodiment the polypeptide includes thesubstitutions K16R, K34R, K40R, T105K and S159K and is conjugated to2-6, typically 3-6 polyethylene glycol moieties with a molecular weightof about 1000-10,000 Da.

[0072] In one embodiment the conjugate of the invention has aglycosylation in T133, i.e. this position is unaltered from thewild-type hG-CSF. This is the natural glycosylation site. Alternatively,the conjugate may be non-glycosylated, although glycosylated conjugatesare preferred.

[0073] In particular, the conjugate may have 2-6, typically 3-6polyethylene glycol moieties with a molecular weight of about 5000-6000Da attached, e.g. mPEG with a molecular weight of about 5 kDa.Preferably, the conjugate has 4-5 polyethylene glycol moieties with amolecular weight of about 5000-6000 Da attached, e.g. 5 kDa mPEG.

[0074] In another embodiment, the conjugate may be produced so as tohave only a single number of PEG moieties attached, e.g. either 2, 3, 4,5 or 6 PEG moieties per polypeptide, or to have a desired mix ofpolypeptide conjugates with different numbers of PEG moieties attached,e.g. a mix having 2-5,2-4, 3-5,3-4, 4-6,4-5 or 5-6 attached PEGmoieties. As indicated above, an example of a preferred conjugate mix isone having 4-5 PEG moieties of about 5 kDa.

[0075] It will be understood that a conjugate having a specific numberof attached PEG moieties, or a mix of conjugates having a defined rangeof numbers of attached PEG moieties, may be obtained by choosingsuitable PEGylation conditions and optionally by using subsequentpurification to separate conjugates having the desired number of PEGmoieties. Examples of methods for separation of G-CSF molecules withdifferent numbers of PEG moieties attached are provided below.Determination of the number of attached PEG moieties may e.g. beperformed using SDS-PAGE. For purposes of the present invention, apolypeptide conjugate may be considered to have a given number ofattached PEG moieties if separation on an SDS-PAGE gel shows no or onlyinsignificant bands other than the band(s) corresponding to the givennumber(s) of PEG moieties. For example, a sample of a polypeptideconjugate is considered to have 4-5 attached PEG groups if an SDS-PAGEgel on which the sample has been run shows bands corresponding to 4 and5 PEG groups, respectively, and only insignificant bands or, preferably,no bands corresponding to 3 or 6 PEG groups.

[0076] While the non-polypeptide moiety of the conjugate according tothis aspect of the invention may be any molecule which, when using thegiven conjugation method has lysine as an attachment group such as acarbohydrate moiety, it is preferred that the non-polypeptide moiety isa polymer molecule. The polymer molecule may be any of the moleculesmentioned in the section entitled “Conjugation to a polymer molecule”,but is preferably selected from the group consisting of linear orbranched polyethylene glycol or another polyalkylene oxide. Preferredpolymer molecules are e.g. mPEG-SPA (in particular SPA-mPEG 5000) fromShearwater Corp. or oxycarbonyl-oxy-N-dicarboxyimide PEG (U.S. Pat. No.5,122,614).

[0077] It will be understood that any of the amino acid changes, inparticular substitutions, specified in this section can be combined withany of the amino acid changes, preferably substitutions, specified inthe other sections herein disclosing specific amino acid modifications,including introduction and/or removal of glycosylation sites.

[0078] Conjugate of the Invention, Wherein the Non-Polypeptide Moiety isa Molecule which has Cysteine as an Attachment Group

[0079] In another aspect the invention relates to a conjugate comprising

[0080] i) a polypeptide exhibiting G-CSF activity, which comprises anamino acid sequence that differs from the amino acid sequence of hG-CSFshown in SEQ ID NO: 1 in at least one substitution selected from thegroup consisting of T1C, P2C, L3C, G4C, P5C, A6C, S7C, S8C, L9C, P10C,Q11C, S12C, F13C, L14C, L15C, E19C, Q20C, V21C, R22C, Q25C, G26C, D27C,A29C, A30C, E33C, A37C, T38C, Y39C, L41C, H43C, P44C, E45C, E46C, V48C,L49C, L50C, H₅₂C, S53C, L54C, I56C, P57C, P60C, L61C, S62C, S63C, P65C,S66C, Q67C, A68C, L69C, Q70C, L71C, A72C, G73C, S76C, Q77C, L78C, S80C,F83C, Q86C, G87C, Q90C, E93C, G94C, S96C, P97C, E98C, L99C, G100C,P101C, T102C, D104C, T105C, Q107C, L108C, D109C, A111C, D112C, F113C,T115C, T116C, W118C, Q119C, Q120C, M121C, E122C, E123C, L124C, M126C,A127C, P128C, A129C, L130C, Q131C, P132C, T133C, Q134C, G135C, A136C,M137C, P138C, A139C, A141C, S142C, A143C, F144C, Q145C, R146C, R147C,S155C, H156C, Q158C, S159C, L161C, E162C, V163C, S164C, Y165C, R166C,V167C, L168C, R169C, H170C, L171C, A172C, Q173C and P174C, and

[0081] ii) at least one non-polypeptide moiety attached to a cysteineresidue of the polypeptide.

[0082] The receptor-binding domain of hG-CSF contains a cysteine residuein position 17 which does not take part in a cystine and which mayadvantageously be removed in order to avoid conjugation of anon-polypeptide moiety to said cysteine. Accordingly, in another, morepreferred aspect the invention relates to a conjugate comprising

[0083] i) a polypeptide exhibiting G-CSF activity, which comprises anamino acid sequence that differs from the amino acid sequence shown inSEQ ID NO: 1 in at least one substitution selected from the groupconsisting of T1C, P2C, L3C, G4C, P5C, A6C, S7C, S8C, L9C, P10C, Q11C,S12C, F13C, L14C, L15C, E19C, Q20C, V21C, R22C, Q25C, G26C, D27C, A29C,A30C, E33C, A37C, T38C, Y39C, L41C, H₄₃C, P44C, E45C, E46C, V48C, L49C,L50C, H₅₂C, S53C, L54C, 156C, P57C, P60C, L61C, S62C, S63C, P65C, S66C,Q67C, A68C, L69C, Q70C, L71C, A72C, G73C, S76C, Q77C, L78C, S80C, F83C,Q86C, G87C, Q90C, E93C, G94C, S96C, P97C, E98C, L99C, G100C, P101C,T102C, D104C, T105C, Q107C, L108C, D109C, A111C, D112C, F113C, T115C,T116C, W118C, Q119C, Q120C, M121C, E122C, E123C, L124C, M126C, A127C,P128C, A129C, L130C, Q131C, P132C, T133C, Q134C, G135C, A136C, M137C,P138C, A139C, A141C, S142C, A143C, F144C, Q145C, R146C, R147C, S155C,H156C, Q158C, S159C, L161C, E162C, V163C, S164C, Y165C, R166C, V167C,L168C, R169C, H170C, L171C, A172C, Q173C and P174C, in combination withremoval of C17, preferably substitution of C17 with any other amino acidresidue, e.g. with a serine residue, and

[0084] ii) a non-polypeptide moiety which has a cysteine residue as anattachment group.

[0085] Preferred substitutions according to this aspect of the inventionare substitutions of arginine with cysteine, for example one or more ofR146C, R147C, R166C and R169C.

[0086] It will be understood that any of the amino acid modifications,in particular substitutions, specified in this section can be combinedwith any of the amino acid changes, in particular substitutions,specified in the other sections herein disclosing specific amino acidmodifications, including introduction and/or removal of glycosylationsites.

[0087] Conjugate of the Invention Wherein the Non-Polypeptide MoietyBinds to an Acid Group or the C-terminal Amino Acid Residue

[0088] In a still further aspect the invention relates to a conjugatecomprising

[0089] i) a polypeptide exhibiting G-CSF activity, which comprises anamino acid sequence that differs from the amino acid sequence shown inSEQ ID NO: 1 in at least one substitution selected from the groupconsisting of T1D, P2D, L3D, G4D, P5D, A6D, S7D, S8D, L9D, P10D, Q11D,S12D, F13D, L14D, L15D, K16D, Q20D, V21D, R22D, K23D, Q25D, G26D, A29D,A30D, K34D, A37D, T38D, Y39D, K40D, L41D, H43D, P44D, V48D, L49D, L50D,H52D, S53D, L54D, 156D, P57D, P60D, L61D, S62D, S63D, P65D, S66D, Q67D,A68D, L69D, Q70D, L71D, A72D, G73D, S76D, Q77D, L78D, S80D, F83D, Q86D,G87D, Q90D, G94D, S96D, P97D, L99D, G100D, P101D, T102D, T105D, Q107D,L108D, A111D, F113D, T115D, T116D, W118D, Q119D, Q120D, M121D, L124D,M126D, A127D, P128D, A129D, L130D, Q131D, P132D, T133D, Q134D, G135D,A136D, M137D, P138D, A139D, A141D, S142D, A143D, F144D, Q145D, R146D,R147D, S155D, H156D, Q158D, S159D, L161D, V163D, S164D, Y165D, R166D,V167D, L168D, R169D, H170D, L171D, A172D, Q173D and P174D; or at leastone substitution selected from the group consisting of T1E, P2E, L3E,G4E, P5E, A6E, S7E, S8E, L9E, P10E, Q11E, S12E, F13E, L14E, L15E, K16E,Q20E, V21E, R22E, K23E, Q25E, G26E, A29E, A30E, K34E, A37E, T38E, Y39E,K40E, L41E, H43E, P44E, V48E, L49E, L50E, H52E, S53E, L54E, 156E, P57E,P60E, L61E, S62E, S63E, P65E, S66E, Q67E, A68E, L69E, Q70E, L71E, A72E,G73E, S76E, Q77E, L78E, S80E, F83E, Q86E, G87E, Q90E, G94E, S96E, P97E,L99E, G100E, P101E, T102E, T105E, Q107E, L108E, A111E, F113E, T115E,T116E, W118E, Q119E, Q120E, M121E, L124E, M126E, A127E, P128E, A129E,L130E, Q131E, P132E, T133E, Q134E, G135E, A136E, M137E, P138E, A139E,A141E, S142E, A143E, F144E, Q145E, R146E, R147E, S155E, H156E, Q158E,S159E, L161E, V163E, S164E, Y165E, R166E, V167E, L168E, R169E, H170E,L171E, A172E, Q173E and P174E; and

[0090] ii) a non-polypeptide moiety having an aspartic acid or aglutamic acid residue as an attachment group.

[0091] Examples of preferred substitutions according to this aspect ofthe invention include Q67D/E, Q70D/E, Q77D/E, Q86D/E, Q90D/E, Q120D/E,Q131D/E, Q134D/E, Q145D/E and Q173D/E.

[0092] In addition to the above listed substitutions, the polypeptide ofthe conjugate according to any of the above aspects may compriseremoval, preferably by substitution, of at least one of the amino acidresidues selected from the group consisting of D27, D104, D109, DI 12,E19, E33, E45, E46, E93, E98, E122, E123, and E163. The substitution maybe for any other amino acid residue, in particular for an asparagine ora glutamine residue, whereby conjugation of these residues can beavoided. In particular, the polypeptide may comprise at least one of thefollowing substitutions: D27N, D104N, D109N, D112N, E19Q, E33Q, E45Q,E46Q, E93Q, E98Q, E122Q, E123Q and E163Q. Preferably, the amino acidsubstitution in one or more of the above positions may in addition becombined with at least one of the following substitutions: D109N, D112N,E19Q, E122Q and E123Q.

[0093] While the non-polypeptide moiety of the conjugate according tothis aspect of the invention, which has an acid group as an attachmentgroup, can be any non-polypeptide moiety with such property, it ispresently preferred that the non-polypeptide moiety is a polymermolecule or an organic derivatizing agent, in particular a polymermolecule, and the conjugate is prepared e.g. as described by Sakane andPardridge, Pharmaceutical Research, Vol. 14, No. 8, 1997, pp 1085-1091.

[0094] It will be understood that any of the amino acid changes, inparticular substitutions, specified in this section can be combined withany of the amino acid changes, in particular substitutions specified inthe other sections herein disclosing specific amino acid changes,including introduction and/or removal of glycosylation sites.

[0095] Other Conjugates of the Invention

[0096] In addition to the non-polypeptide moieties specified above e.g.in the sections entitled “Conjugate of the invention . . . ” theconjugate of the invention may contain one or more carbohydrate moietiesas a consequence of the polypeptide being expressed in a glycosylatinghost cell to result in glycosylation at the natural glycosylation siteof hG-CSF (T133) and/or at introduced glycosylation site(s).

[0097] Conjugate of the Invention Wherein the Non-Polypeptide Moiety isa Carbohydrate Moiety

[0098] In a further aspect the invention relates to a conjugatecomprising a glycosylated polypeptide exhibiting G-CSF activity, whichcomprises an amino acid sequence that differs from that shown in SEQ IDNO:1 in that at least one non-naturally occurring glycosylation site hasbeen introduced into the amino acid sequence by way of at least onesubstitution selected from the group consisting of L3N+P5S/T, P5N, A6N,S8N+P10S/T, P10N, Q11N+F13S/T, S12N+L14S/T, F13N+L15S/T, L14N+K16S/T,K16N+L18S/T, E19N+V21S/T, Q20N+R22S/T, V21N+K23S/T, R22N+124S/T,K23N+Q25S/T, Q25N+D27S/T, G26N+G28S/T, D27N+A29S/T, A29N+L31S/T,A30N+Q32S/T, E33N+L35S/T, A37N+Y39S/T, T38N+K40S/T, Y39N+L41S/T,P44N+E46S/T, E45N+L47S/T, E46N+V48S/T, V48N+L50S/T, L49N+G51S/T,L50N+H52S/T, H₅₂N+L54S/T, S53N+G55S/T, P60N, L61N, S63N+P65S/T,P65N+Q67S/T, S66N+A68S/T, Q67N+L69S/T, A68N+Q70S/T, L69N+L71S/T,Q70N+A72S/T, L71N+G73S/T, G73N+L75S/T, S76N+L78S/T, Q77N+H79S/T, L78N,S80N+L82S/T, F83N+Y85S/T, Q86N+L88S/T, G87N+L89S/T, Q90N+L92S/T,E93N+195S/T, P97N+L99S/T, L99N+P101S/T, P101N+L103S/T, T102N+D104S/T,D104N+L106S/T, T105N+Q107S/T, Q107N+D109S/T, L108N+VS/T, D109N+A111S/T,A111N+F113S/T, D112N+A114S/T, F113N, T115N+I117S/T, T116N+W118S/T,W118N+Q120S/T, Q119N+M121S/T, Q120N+E122S/T, M121N+E123S/T,E122N+L124S/T, E123N+G125S/T, L124N+M126S/T, M126N+P128S/T,P128N+L130S/T, L130N+P132S/T, P132N+Q134S/T, T133N+G135S/T,Q134N+A136S/T, A136N+P138S/T, P138N+F140S/T, A139N+A141S/T,A141N+A143S/T, S142N+F144S/T, A143N+Q145S/T, F144N+R146S/T,Q145N+R147S/T, R146N+A148S/T, R147N+G149S/T, S155N+L157S/T,H156N+Q158S/T, S159N+L161S/T, L161N+V163S/T, E162N, V163N+Y165S/T,S164N+R166S/T, Y165N+V167S/T, R166N+L168S/T, V167N+R169S/T,L168N+H170S/T, R169N+L171S/T and H170N+A172S/T, wherein S/T indicates anS or a T residue, preferably a T residue.

[0099] It will be understood that in order to prepare a conjugateaccording to this aspect the polypeptide must be expressed in aglycosylating host cell capable of attaching oligosaccharide moieties atthe glycosylation site(s) or alternatively subjected to in vitroglycosylation. Examples of glycosylating host cells are given in thesection further below entitled “Coupling to an oligosaccharide moiety”.

[0100] Alternatively, the conjugate according to this aspect comprises apolypeptide exhibiting G-CSF activity, which comprises an amino acidsequence that differs from that shown in SEQ ID NO: 1 in at least onesubstitution selected from the group consisting of P5N, A6N, P10N, P60N,L61N, L78N, F113N and E162N, in particular from the group consisting ofP5N, A6N, P10N, P60N, L61N, F113N and E162N, such as from the groupconsisting of P60N, L61N, F113N and E162N.

[0101] Alternatively, the conjugate according to this aspect comprises apolypeptide exhibiting G-CSF activity, which comprises an amino acidsequence that differs from that shown in SEQ ID NO:1 in at least onesubstitution selected from the group consisting of D27N+A29S, D27N+A29T,D104N+L106S, D104N+L106T, D109N+A111S, D109N+A111T, D112N+A114S andD112N+A114T, more preferably from the group consisting of D27N+A29S,D27N+A29T, D104N+L106S, D104N+L106T, D112N+A114S and D112N+A114T, suchas from the group consisting of D27N+A29S, D27N+A29T, D104N+L106S andD104N+L106T.

[0102] In addition to a carbohydrate molecule, the conjugate accordingto the aspect of the invention described in the present section maycontain additional non-polypeptide moieties, in particular a polymermolecule, as described in the present application, conjugated to one ormore attachment groups present in the polypeptide part of the conjugate.

[0103] It will be understood that any of the amino acid changes, inparticular substitutions, specified in this section can be combined withany of the amino acid changes, in particular substitutions, specified inthe other sections herein disclosing specific amino acid changes.

[0104] Circularly Permuted Variants

[0105] In a further embodiment, the polypeptide part of the polypeptideconjugate of the invention may be in the form of a circularly permutedvariant of a polypeptide sequence otherwise disclosed herein. In such acircularly permuted polypeptide, the original N-terminus and C-terminusare joined together either directly by a peptide bond or indirectly viaa peptide linker, while new N- and C-termini are formed between twoadjacent amino acid residues that originally were joined by a peptidebond. Since the original N- and C-termini will normally be located atsome distance from each other, they will typically be linked by means ofa peptide linker having a suitable length and composition so that thestructure and activity of the conjugate is not adversely affected. Itwill be clear that the new N-terminus and C-terminus should not beformed between an amino acid residue pair where this would interferewith the activity of the polypeptide. Circularly permuted G-CSF receptoragonists are disclosed in U.S. Pat. No. 6,100,070, to which reference ismade for further information on selecting peptide linkers and thelocation of the new N-terminus and C-terminus as well as methods forproducing them such variants.

[0106] White Blood Cell and Neutrophil Formation of Conjugates of theInvention

[0107] In a further embodiment, the polypeptide conjugate of theinvention may be characterized as being a conjugate exhibiting G-CSFactivity and comprising a polypeptide with an amino acid sequence thatdiffers in at least one amino acid residue from the amino acid sequenceshown in SEQ ID NO: 1 and having at least one non-polypeptide moietyattached to an attachment group of the polypeptide, the polypeptideconjugate further fulfilling at least one of the following criteria(A)-(D):

[0108] (A) after one subcutaneous administration of 100 microgram per kgbody weight to rats (based on the weight of the polypeptide part of theconjugate) it:

[0109] i) increases formation of white blood cells with at least aboutthe same rate and to at least about the same level (measured as numberof cells per liter of blood) as administration of 100 microgram ofnon-conjugated hG-CSF per kg body weight for a period of 6 hours,preferably 12 hours after administration, and

[0110] ii) increases the level of white blood cells (measured as numberof cells per liter blood) above the level of white blood cells prior toadministration for a period of at least about 96 hours, preferably forat least about 120 hours;

[0111] (B) after one subcutaneous administration of 25 microgram per kgbody weight to rats (based on the weight of the polypeptide part of theconjugate) it:

[0112] i) increases formation of white blood cells with at least aboutthe same rate and to at least about the same level (measured as numberof cells per liter of blood) as administration of 100 microgram ofnon-conjugated hG-CSF per kg body weight for a period of 6 hours,preferably 12 hours after administration, and

[0113] ii) increases the level of white blood cells (measured as numberof cells per liter blood) above the level of white blood cells prior toadministration for a period of at least about 72 hours, preferably atleast about 96 hours, more preferably at least about 120 hours;

[0114] (C) after one subcutaneous administration of 100 microgram per kgbody weight to rats (based on the weight of the polypeptide part of theconjugate) it:

[0115] i) increases formation of neutrophils with at least about thesame rate and to at least about the same level (measured as number ofcells per liter of blood) as administration of 100 microgram ofnon-conjugated hG-CSF per kg body weight for a period of 6 hours,preferably 12 hours after administration, and

[0116] ii) increases the level of neutrophils (measured as number ofcells per liter blood) above the level of neutrophils prior toadministration for a period of at least about 96 hours, preferably atleast about 120 hours;

[0117] (D) after one subcutaneous administration of 25 microgram per kgbody weight to rats (based on the weight of the polypeptide part of theconjugate) it:

[0118] i) increases formation of neutrophils with at least about thesame rate and to at least about the same level (measured as number ofcells per liter of blood) as administration of 100 microgram ofnon-conjugated hG-CSF per kg body weight for a period of 6 hours,preferably 12 hours after administration, and

[0119] ii) increases the level of neutrophils (measured as number ofcells per liter blood) above the level of neutrophils prior toadministration for a period of at least about 72 hours, preferably atleast about 96 hours, more preferably at least about 120 hours.

[0120] Non-Polypeptide Moiety of the Conjugate of the Invention

[0121] As indicated further above the non-polypeptide moiety of theconjugate of the invention is preferably selected from the groupconsisting of a polymer molecule, a lipophilic compound, a carbohydratemoiety (e.g. by way of in vivo glycosylation) and an organicderivatizing agent. All of these agents may confer desirable propertiesto the polypeptide part of the conjugate, in particular increasedfunctional in vivo half-life and/or increased serum half-life. Thepolypeptide part of the conjugate is normally conjugated to only onetype of non-polypeptide moiety, but may also be conjugated to two ormore different types of non-polypeptide moieties, e.g. to a polymermolecule and an oligosaccharide moiety, to a lipophilic group and anoligosaccharide moiety, to an organic derivatizing agent and anoligosaccharide moiety, to a lipophilic group and a polymer molecule,etc. The conjugation to two or more different non-polypeptide moietiesmay be done simultaneously or sequentially.

[0122] Methods for Preparing a Conjugate of the Invention

[0123] In the following sections “Conjugation to a lipophilic compound”,“Conjugation to a polymer molecule”, “Conjugation to an oligosaccharidemoiety” and “Conjugation to an organic derivatizing agent” conjugationto specific types of non-polypeptide moieties is described. In general,a polypeptide conjugate according to the invention may be produced byculturing an appropriate host cell under conditions conducive forexpression of the polypeptide, and recovering the polypeptide, whereina) the polypeptide comprises at least one N- or O-glycosylation site andthe host cell is a eukaryotic host cell capable of in vivoglycosylation, and/or b) the polypeptide is subjected to conjugation toa non-polypeptide moiety in vitro.

[0124] Conjugation to a Lipophilic Compound

[0125] The polypeptide and the lipophilic compound may be conjugated toeach other, either directly or by use of a linker. The lipophiliccompound may be a natural compound such as a saturated or unsaturatedfatty acid, a fatty acid diketone, a terpene, a prostaglandin, avitamin, a carotenoid or steroid, or a synthetic compound such as acarbon acid, an alcohol, an amine and sulphonic acid with one or morealkyl, aryl, alkenyl or other multiple unsaturated compounds. Theconjugation between the polypeptide and the lipophilic compound,optionally through a linker, may be done according to methods known inthe art, e.g. as described by Bodanszky in Peptide Synthesis, JohnWiley, New York, 1976 and in WO 96/12505.

[0126] Conjugation to a Polymer Molecule

[0127] The polymer molecule to be coupled to the polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror heteropolymer, typically with a molecular weight in the range ofabout 300-100,000 Da, such as about 500-20,000 Da, more preferably inthe range of about 1000-15,000 Da, even more preferably in the range ofabout 2000-12,000 Da, such as about 3000-10,000. When used about polymermolecules herein, the word “about” indicates an approximate averagemolecular weight and reflects the fact that there will normally be acertain molecular weight distribution in a given polymer preparation.

[0128] Examples of homo-polymers include a polyol (i.e. poly-OH), apolyamine (i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer which comprises different coupling groups,such as a hydroxyl group and an amine group.

[0129] Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as linear or branchedpolyethylene glycol (PEG) and polypropylene glycol (PPG), poly-vinylalcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone),polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, dextran, including carboxymethyl-dextran, or any otherbiopolymer suitable for reducing immunogenicity and/or increasingfunctional in vivo half-life and/or serum half-life. Another example ofa polymer molecule is human albumin or another abundant plasma protein.Generally, polyalkylene glycol-derived polymers are biocompatible,non-toxic, non-antigenic, non-immunogenic, have various water solubilityproperties, and are easily excreted from living organisms.

[0130] PEG is the preferred polymer molecule, since it has only fewreactive groups capable of cross-linking compared to polysaccharidessuch as dextran. In particular, monofunctional PEG, e.g.methoxypolyethylene glycol (mPEG), is of interest since its couplingchemistry is relatively simple (only one reactive group is available forconjugating with attachment groups on the polypeptide). Consequently,the risk of cross-linking is eliminated, the resulting polypeptideconjugates are more homogeneous and the reaction of the polymermolecules with the polypeptide is easier to control.

[0131] To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the hydroxyl end groups of the polymer molecule areprovided in activated form, i.e. with reactive functional groups.Suitable activated polymer molecules are commercially available, e.g.from Shearwater Corp., Huntsville, Ala., USA, or from PolyMASCPharmaceuticals plc, UK. Alternatively, the polymer molecules can beactivated by conventional methods known in the art, e.g. as disclosed inWO 90/13540. Specific examples of activated linear or branched polymermolecules for use in the present invention are described in theShearwater Corp. 1997 and 2000 Catalogs (Functionalized BiocompatiblePolymers for Research and pharmaceuticals, Polyethylene Glycol andDerivatives, incorporated herein by reference). Specific examples ofactivated PEG polymers include the following linear PEGs: NHS-PEG (e.g.SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, andSCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG,ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs suchas PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 and U.S. Pat.No. 5,643,575, both of which are incorporated herein by reference.Furthermore, the following publications, incorporated herein byreference, disclose useful polymer molecules and/or PEGylationchemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,476,653, WO97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No.5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28024, WO 95/00162, WO 95/11924, WO 95/13090, WO 95/33490, WO96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No.5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673,EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400 472, EP 183 503and EP 154 316.

[0132] The conjugation of the polypeptide and the activated polymermolecules is conducted by use of any conventional method, e.g. asdescribed in the following references (which also describe suitablemethods for activation of polymer molecules): R. F. Taylor, (1991),“Protein immobilisation. Fundamental and applications”, Marcel Dekker,N.Y.; S. S. Wong, (1992), “Chemistry of Protein Conjugation andCrosslinking”, CRC Press, Boca Raton; G. T. Hermanson et al., (1993),“Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.). Theskilled person will be aware that the activation method and/orconjugation chemistry to be used depends on the attachment group(s) ofthe polypeptide (examples of which are given further above), as well asthe functional groups of the polymer (e.g. being amine, hydroxyl,carboxyl, aldehyde, sulfydryl, succinimidyl, maleimide, vinysulfone orhaloacetate). The PEGylation may be directed towards conjugation to allavailable attachment groups on the polypeptide (i.e. such attachmentgroups that are exposed at the surface of the polypeptide) or may bedirected towards one or more specific attachment groups, e.g. theN-terminal amino group (U.S. Pat. No. 5,985,265). Furthermore, theconjugation may be achieved in one step or in a stepwise manner (e.g. asdescribed in WO 99/55377).

[0133] It will be understood that the PEGylation is designed so as toproduce the optimal molecule with respect to the number of PEG moleculesattached, the size and form of such molecules (e.g. whether they arelinear or branched), and where in the polypeptide such molecules areattached. The molecular weight of the polymer to be used will be chosentaking into consideration the desired effect to be achieved. Forinstance, if the primary purpose of the conjugation is to achieve aconjugate having a high molecular weight and larger size (e.g. to reducerenal clearance), one may choose to conjugate either one or a few highmolecular weight polymer molecules or a number of polymer molecules witha smaller molecular weight to obtain the desired effect. Preferably,however, several polymer molecules with a lower molecular weight will beused. This is also the case if a high degree of epitope shielding isdesired. In such cases, 2-8 polymers with a molecular weight of e.g.about 5,000 Da, such as 3-6 such polymers, may for example be used. Asthe examples below illustrate, it may be advantageous to have a largernumber of polymer molecules with a lower molecular weight (e.g. 4-6 witha MW of 5000) compared to a smaller number of polymer molecules with ahigher molecular weight (e.g. 1-3 with a MW of 12,000-20,000) in termsof improving the functional in vivo half-life of the polypeptideconjugate, even where the total molecular weight of the attached polymermolecules in the two cases is the same or similar. It is believed thatthe presence of a larger number of smaller polymer molecules providesthe polypeptide with a larger diameter or apparent size than e.g. asingle yet larger polymer molecule, at least when the polymer moleculesare relatively uniformly distributed on the polypeptide surface.

[0134] It has further been found that advantageous results are obtainedwhen the apparent size (also referred to as the “apparent molecularweight” or “apparent mass”) of at least a major portion of the conjugateof the invention is at least about 50 kDa, preferably at least about 55kDa, more preferably at least about 60 kDa, e.g. at least about 66 kDa.This is believed to be due to the fact that renal clearance issubstantially eliminated for conjugates having a sufficiently largeapparent size. In the present context, the “apparent size” of a G-CSFconjugate or polypeptide is determined by the SDS-PAGE method describedin the examples section below.

[0135] The use of the term “major portion” is related to the fact thatthe polypeptide conjugates of the invention will typically compriseindividual conjugates having varying numbers of non-polypeptide moietiesattached. For example, a given polypeptide subjected to PEGylation undera given set of PEGylation conditions may result in a composition inwhich most of the individual polypeptide conjugates have e.g. between 3and 5 PEG groups attached, with a majority of the conjugates having 4PEG groups attached. It will be clear that the apparent molecular weightof these individual conjugate molecules will vary. In this example, ifwe assume that a G-CSF polypeptide is conjugated to PEG groups with a MWof 5 kDa, conjugates having only 3 PEG groups attached will be seen onan SDS-PAGE gel as a band that is likely to have an apparent molecularweight of less than about 50 kDa, while conjugates having 4 or 5 PEGgroups attached will result in bands with progressively higher apparentmolecular weights that most likely all are greater than about 50 kDa.Thus, in this example there would be 3 major bands on an SDS-PAGE gel,corresponding to conjugates with 3, 4 or 5 attached PEG groups,respectively. The term “major portion” in the context of the presentspecification and claims is therefore intended to refer to the fact thatat least one of these major bands on an SDS-PAGE gel will correspond tothe indicated minimum apparent molecular weight.

[0136] Preferably, at least 50% of the individual conjugate moleculeswill have a minimum apparent size as described above. More preferably,at least 60% of the individual conjugate molecules with have such aminimum apparent size, still more preferably at least 70%, 75%, 80% or85%. Most preferably, at least 90% of the individual conjugate moleculeswill have a minimum apparent size as described above, i.e. at least 50kDa and preferably higher, such as at least 55 kDa or 60 kDa.

[0137] It will be understood that the apparent size in kDa of aconjugate or polypeptide is not necessarily the same as the actualmolecular weight of the conjugate or polypeptide. Rather, the apparentsize is a reflection of both the actual molecular weight and the overallbulk. Since, in most cases, attachment of one or more PEG groups orother non-polypeptide moieties will result in a relatively largeincrease of the bulk of the polypeptide to which such moieties areattached, the polypeptide conjugates of the invention will normally havean apparent size that exceeds the actual molecular weight of theconjugate. Therefore, in connection with renal clearance, a conjugate ofthe invention can easily exhibit properties characteristic of a ispolypeptide with a molecular weight above e.g. 66 kDa (corresponding tothe apparent size) but have an actual molecular weight well below 66kDa. This effect on apparent size is believed to be responsible for theobservation that attachment of, for example, four PEG groups each havinga molecular weight of 5 kDa provides results that are superior to acorresponding polypeptide with a single 20 kDa PEG group attached.

[0138] While conjugation of only a single polymer molecule to a singleattachment group on the protein is not preferred, in the event that onlyone polymer molecule is attached, it will generally be advantageous thatthe polymer molecule, which may be linear or branched, has a relativelyhigh molecular weight, e.g. about 20 kDa.

[0139] In a further preferred embodiment, the conjugates of theinvention have 1) at least a major portion with an apparent molecularweight of at least about 50 kDa and 2) a reduced in vitro bioactivity(reduced receptor binding affinity) compared to hG-CSF as describedabove. It has been found that such conjugates have both a low renalclearance as a result of the large apparent size and a lowreceptor-mediated clearance as a result of the low in vitro bioactivity(low receptor binding affinity). The overall result is excellentperformance in terms of effective stimulation of neutrophils togetherwith a significantly increased in vivo half-life and thus a longduration of action that provides important clinical advantages.

[0140] Normally, the polymer conjugation is performed under conditionsaiming at reacting as many of the available polymer attachment groups aspossible with polymer molecules. This is achieved by means of a suitablemolar excess of the polymer in relation to the polypeptide (number ofattachment sites). Typical molar ratios of activated polymer moleculesto polypeptide attachment sites are up to about 1000-1, such as up toabout 200-1 or up to about 100-1. In some cases, the ratio may besomewhat lower, however, such as up to about 50-1, 10-1 or 5-1, e.g. ifa lower degree of polymer attachment is desired.

[0141] It is also contemplated according to the invention to couple thepolymer molecules to the polypeptide through a linker. Suitable linkersare well known to the skilled person. A preferred example is cyanuricchloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581;U.S. Pat. No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym.Chem. Ed., 24, 375-378.

[0142] Subsequent to the conjugation residual activated polymermolecules are blocked according to methods known in the art, e.g. byaddition of primary amine to the reaction mixture, and the resultinginactivated polymer molecules are removed by a suitable method (seeMaterials and Methods).

[0143] In a preferred embodiment, the polypeptide conjugate of theinvention comprises a PEG molecule attached to some, most or preferablysubstantially all of the lysine residues in the polypeptide availablefor PEGylation, in particular a linear or branched PEG molecule, e.g.with a molecular weight of about 1-15 kDa, typically about 2-12 kDa,such as about 3-10 kDa, e.g. about 5 or 6 kDa.

[0144] It will be understood that depending on the circumstances, e.g.the amino acid sequence of the polypeptide, the nature of the activatedPEG compound being used and the specific PEGylation conditions,including the molar ratio of PEG to polypeptide, varying degrees ofPEGylation may be obtained, with a higher degree of PEGylation generallybeing obtained with a higher ratio of PEG to polypeptide. The PEGylatedpolypeptides resulting from any given PEGylation process will, however,normally comprise a stochastic distribution of polypeptide conjugateshaving slightly different degrees of PEGylation. If desired, such amixture of polypeptide species having different numbers of PEG moietiesattached may be subjected to purification, e.g. using the methodsdescribed in the examples below, to obtain a product having a moreuniform degree of PEGylation.

[0145] In yet another embodiment, the polypeptide conjugate of theinvention may comprise a PEG molecule attached to the lysine residues inthe polypeptide available for PEGylation, and in addition to theN-terminal amino acid residue of the polypeptide.

[0146] Coupling to an Oligosaccharide Moiety

[0147] The conjugation to an oligosaccharide moiety may take place invivo or in vitro. In order to achieve in vivo glycosylation of a G-CSFmolecule comprising one or more glycosylation sites the nucleotidesequence encoding the polypeptide must be inserted in a glycosylating,eukaryotic expression host. The expression host cell may be selectedfrom fungal (filamentous fungal or yeast), insect or animal cells orfrom transgenic plant cells. In one embodiment the host cell is amammalian cell, such as a CHO cell, BHK or HEK, e.g. HEK 293, cell, oran insect cell, such as an SF9 cell, or a yeast cell, e.g. S. cerevisiaeor Pichia pastoris, or any of the host cells mentioned hereinafter.Covalent in vitro coupling of glycosides (such as dextran) to amino acidresidues of the polypeptide may also be used, e.g. as described in WO87/05330 and in Aplin et al., CRC Crit Rev. Biochem., pp. 259-306, 1981.

[0148] The in vitro coupling of oligosaccharide moieties or PEG toprotein- and peptide-bound Gln-residues can be carried out bytransglutaminases (TG'ases). Transglutaminases catalyze the transfer ofdonor amine-groups to protein- and peptide-bound Gln-residues in aso-called cross-linking reaction. The donor-amine groups can be protein-or peptide-bound e.g. as the c-amino-group in Lys-residues or can bepart of a small or large organic molecule. An example of a small organicmolecule functioning as an amino-donor in TG'ase-catalyzed cross-linkingis putrescine (1,4-diaminobutane). An example of a larger organicmolecule functioning as an amino-donor in TG'ase-catalyzed cross-linkingis an amine-containing PEG (Sato et al., Biochemistry 35, 13072-13080).

[0149] TG'ases are in general highly specific enzymes, and not everyGln-residue exposed on the surface of a protein is accessible toTG'ase-catalyzed cross-linking to amino-containing substances. On thecontrary, only a few Gln-residues function naturally as TG'asesubstrates, but the exact parameters governing which Gln-residues aregood TG'ase substrates remain unknown. Thus, in order to render aprotein susceptible to TG'ase-catalyzed cross-linking reactions it isoften a prerequisite to add at convenient positions stretches of aminoacid sequence known to function very well as TG'ase substrates. Severalamino acid sequences are known to be or to contain excellent naturalTG'ase substrates e.g. substance P, elafin, fibrinogen, fibronectin,α₂-plasmin inhibitor, α-caseins, and β-caseins.

[0150] Coupling to an Organic Derivatizing Agent

[0151] Covalent modification of the polypeptide exhibiting G-CSFactivity may be performed by reacting one or more attachment groups ofthe polypeptide with an organic derivatizing agent. Suitablederivatizing agents and methods are well known in the art. For example,cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-p-(4-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues are derivatizedby reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent isrelatively specific for the histidyl side chain. Para-bromophenacylbromide is also useful. The reaction is preferably performed in 0.1 Msodium cacodylate at pH 6.0. Lysinyl and amino terminal residues arereacted with succinic or other carboxylic acid anhydrides.Derivatization with these agents has the effect of reversing the chargeof the lysinyl residues. Other suitable reagents for derivatizingα-amino-containing residues include imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione andtransaminase-catalyzed reaction with glyoxylate. Arginyl residues aremodified by reaction with one or several conventional reagents, amongthem phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, andninhydrin. Derivatization of arginine residues requires that thereaction be performed in alkaline conditions because of the high pKa ofthe guanidine functional group.

[0152] Furthermore, these reagents may react with the groups of lysineas well as the arginine guanidino group. Carboxyl side groups (aspartylor glutamyl) are selectively modified by reaction with carbodiimides(R—N═C═N—R′), where R and R′ are different alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

[0153] Blocking of the Functional Site

[0154] It has been reported that excessive polymer conjugation can leadto a loss of activity of the polypeptide to which the polymer isconjugated. This problem can be eliminated by e.g. removal of attachmentgroups located at the functional site or by blocking the functional siteprior to conjugation so that the functional site is blocked duringconjugation. The latter strategy constitutes a further embodiment of theinvention (the first strategy being exemplified further above, e.g. byremoval of lysine residues which may be located close to the functionalsite). More specifically, according to the second strategy theconjugation between the polypeptide and the non-polypeptide moiety isconducted under conditions where the functional site of the polypeptideis blocked by a helper molecule capable of binding to the functionalsite of the polypeptide.

[0155] Preferably, the helper molecule is one which specificallyrecognizes a functional site of the polypeptide, such as a receptor, inparticular the G-CSF receptor or a part of the G-CSF receptor.

[0156] Alternatively, the helper molecule may be an antibody, inparticular a monoclonal antibody recognizing the polypeptide exhibitingG-CSF activity. In particular, the helper molecule may be a neutralizingmonoclonal antibody.

[0157] The polypeptide is allowed to interact with the helper moleculebefore effecting conjugation. This ensures that the functional site ofthe polypeptide is shielded or protected and consequently unavailablefor derivatization by the non-polypeptide moiety such as a polymer.Following its elution from the helper molecule, the conjugate betweenthe non-polypeptide moiety and the polypeptide can be recovered with atleast a partially preserved functional site.

[0158] The subsequent conjugation of the polypeptide having a blockedfunctional site to a polymer, a lipophilic compound, an oligosaccharidemoiety, an organic derivatizing agent or any other compound is conductedin the normal way, e.g. as described in the sections above entitled“Conjugation to . . . ”.

[0159] Irrespective of the nature of the helper molecule to be used toshield the functional site of the polypeptide from conjugation, it isdesirable that the helper molecule is free of or comprises only a fewattachment groups for the non-polypeptide moiety of choice in part(s) ofthe molecule where the conjugation to such groups would hamperdesorption of the conjugated polypeptide from the helper molecule.Hereby, selective conjugation to attachment groups present innon-shielded parts of the polypeptide can be obtained and it is possibleto reuse the helper molecule for repeated cycles of conjugation. Forinstance, if the non-polypeptide moiety is a polymer molecule such asPEG, which has the epsilon amino group of a lysine or N-terminal aminoacid residue as an attachment group, it is desirable that the helpermolecule is substantially free of conjugatable epsilon amino groups,preferably free of any epsilon amino groups. Accordingly, in a preferredembodiment the helper molecule is a protein or peptide capable ofbinding to the functional site of the polypeptide, which protein orpeptide is free of any conjugatable attachment groups for thenon-polypeptide moiety of choice.

[0160] Of particular interest in connection with the embodiment of thepresent invention wherein the polypeptide conjugates are prepared from adiversified population of nucleotide sequences encoding a polypeptide ofinterest, the blocking of the functional group is effected in microtiterplates prior to conjugation, for instance by plating the expressedpolypeptide variant in a microtiter plate containing an immobilizedblocking group such as a receptor, an antibody or the like.

[0161] In a further embodiment the helper molecule is first covalentlylinked to a solid phase such as column packing materials, for instanceSephadex or agarose beads, or a surface, e.g. a reaction vessel.Subsequently, the polypeptide is loaded onto the column materialcarrying the helper molecule and conjugation carried out according tomethods known in the art, e.g. as described in the sections aboveentitled “Conjugation to . . . . ”. This procedure allows thepolypeptide conjugate to be separated from the helper molecule byelution. The polypeptide conjugate is eluted by conventional techniquesunder physico-chemical conditions that do not lead to a substantivedegradation of the polypeptide conjugate. The fluid phase containing thepolypeptide conjugate is separated from the solid phase to which thehelper molecule remains covalently linked. The separation can beachieved in other ways: For instance, the helper molecule may bederivatized with a second molecule (e.g. biotin) that can be recognizedby a specific binder (e.g. streptavidin). The specific binder may belinked to a solid phase, thereby allowing the separation of thepolypeptide conjugate from the helper molecule-second molecule complexthrough passage over a second helper-solid phase column which willretain, upon subsequent elution, the helper molecule-second moleculecomplex, but not the polypeptide conjugate. The polypeptide conjugatemay be released from the helper molecule in any appropriate fashion.Deprotection may be achieved by providing conditions in which the helpermolecule dissociates from the functional site of the G-CSF to which itis bound. For instance, a complex between an antibody to which a polymeris conjugated and an anti-idiotypic antibody can be dissociated byadjusting the pH to an acid or alkaline pH.

[0162] Conjugation of a Tagged Polypeptide

[0163] In an alternative embodiment the polypeptide is expressed as afusion protein with a tag, i.e. an amino acid sequence or peptidestretch made up of typically 1-30, such as 1-20 amino acid residues.Besides allowing for fast and easy purification, the tag is a convenienttool for achieving conjugation between the tagged polypeptide and thenon-polypeptide moiety. In particular, the tag may be used for achievingconjugation in microtiter plates or other carriers, such as paramagneticbeads, to which the tagged polypeptide can be immobilized via the tag.The conjugation to the tagged polypeptide in e.g. microtiter plates hasthe advantage that the tagged polypeptide can be immobilized in themicrotiter plates directly from the culture broth (in principle withoutany purification) and subjected to conjugation. Thereby, the totalnumber of process steps (from expression to conjugation) can be reduced.Furthermore, the tag may function as a spacer molecule, ensuring animproved accessibility to the immobilized polypeptide to be conjugated.The conjugation using a tagged polypeptide may be to any of thenon-polypeptide moieties disclosed herein, e.g. to a polymer moleculesuch as PEG.

[0164] The identity of the specific tag to be used is not critical aslong as the tag is capable of being expressed with the polypeptide andis capable of being immobilized on a suitable surface or carriermaterial. A number of suitable tags are commercially available, e.g.from Unizyme Laboratories, Denmark. For instance, the tag may consist ofany of the following sequences:

[0165] Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-Gln (SEQID NO:5)

[0166] His-His-His-His-His-His (SEQ ID NO:9)

[0167] Met-Lys-His-His-His-His-His-His (SEQ ID NO:10)

[0168] Met-Lys-His-His-Ala-His-His-Gln-His-His (SEQ ID NO:11)

[0169] Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln (SEQ IDNO: 12)

[0170] or any of the following:

[0171] EQKLI SEEDL (SEQ ID NO:13; a C-terminal tag described in Mol.Cell. Biol. 5:3610-16, 1985)

[0172] DYKDDDDK (SEQ ID NO:14; a C- or N-terminal tag)

[0173] YPYDVPDYA (SEQ ID NO: 15)

[0174] Antibodies against the above tags are commercially available,e.g. from ADI, Aves Lab and Research Diagnostics.

[0175] A convenient method for using a tagged polypeptide for PEGylationis given in the Materials and Methods section below. The subsequentcleavage of the tag from the polypeptide may be achieved by use ofcommercially available enzymes.

[0176] Methods for Preparing a Polypeptide of the Invention or thePolypeptide Part of the Conjugate of the Invention

[0177] The polypeptide of the present invention or the polypeptide partof a conjugate of the invention, optionally in glycosylated form, may beproduced by any suitable method known in the art. Such methods includeconstructing a nucleotide sequence encoding the polypeptide andexpressing the sequence in a suitable transformed or transfected host.However, polypeptides of the invention may be produced, albeit lessefficiently, by chemical synthesis or a combination of chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology.

[0178] A nucleotide sequence encoding a polypeptide or the polypeptidepart of a conjugate of the invention may be constructed by isolating orsynthesizing a nucleotide sequence encoding the parent G-CSF, such ashG-CSF with the amino acid sequence shown in SEQ ID NO: 1, and thenchanging the nucleotide sequence so as to effect introduction (i.e.insertion or substitution) or deletion (i.e. removal or substitution) ofthe relevant amino acid residue(s).

[0179] The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence is prepared by chemical synthesis, e.g. by using anoligonucleotide synthesizer, wherein oligonucleotides are designed basedon the amino acid sequence of the desired polypeptide, and preferablyselecting those codons that are favored in the host cell in which therecombinant polypeptide will be produced. For example, several smalloligonucleotides coding for portions of the desired polypeptide may besynthesized and assembled by PCR, ligation or ligation chain reaction(LCR) (Barany, PNAS 88:189-193, 1991). The individual oligonucleotidestypically contain 5′ or 3′ overhangs for complementary assembly.

[0180] Alternative nucleotide sequence modification methods areavailable for producing polypeptide variants for high throughputscreening, for instance methods which involve homologous cross-over suchas disclosed in U.S. Pat. No. 5,093,257, and methods which involve geneshuffling, i.e. recombination between two or more homologous nucleotidesequences resulting in new nucleotide sequences having a number ofnucleotide alterations when compared to the starting nucleotidesequences. Gene shuffling (also known as DNA shuffling) involves one ormore cycles of random fragmentation and reassembly of the nucleotidesequences, followed by screening to select nucleotide sequences encodingpolypeptides with desired properties. In order for homology-basednucleic acid shuffling to take place, the relevant parts of thenucleotide sequences are preferably at least 50% identical, such as atleast 60% identical, more preferably at least 70% identical, such as atleast 80% identical. The recombination can be performed in vitro or invivo.

[0181] Examples of suitable in vitro gene shuffling methods aredisclosed by Stemmer et al. (1994), Proc. Natl. Acad. Sci. USA; vol. 91,pp. 10747-10751; Stemmer (1994), Nature, vol. 370, pp. 389-391; Smith(1994), Nature vol. 370, pp. 324-325; Zhao et al., Nat. Biotechnol.1998, Mar; 16(3): 258-61; Zhao H. and Arnold, F B, Nucleic AcidsResearch, 1997, Vol. 25. No. 6 pp. 1307-1308; Shao et al., Nucleic AcidsResearch 1998, Jan 15; 26(2): pp. 681-83; and WO 95/17413. An example ofa suitable in vivo shuffling method is disclosed in WO 97/07205. Othertechniques for mutagenesis of nucleic acid sequences by in vitro or invivo recombination are disclosed e.g. in WO 97/20078 and U.S. Pat. No.5,837,458. Examples of specific shuffling techniques include “familyshuffling”, “synthetic shuffling” and “in silico shuffling”. Familyshuffling involves subjecting a family of homologous genes fromdifferent species to one or more cycles of shuffling and subsequentscreening or selection. Family shuffling techniques are disclosed e.g.by Crameri et al. (1998), Nature, vol. 391, pp. 288-291; Christians etal. (1999), Nature Biotechnology, vol. 17, pp. 259-264; Chang et al.(1999), Nature Biotechnology, vol. 17, pp. 793-797; and Ness et al.(1999), Nature Biotechnology, vol. 17, 893-896. Synthetic shufflinginvolves providing libraries of overlapping synthetic oligonucleotidesbased e.g. on a sequence, alignment of homologous genes of interest. Thesynthetically generated oligonucleotides are recombined, and theresulting recombinant nucleic acid sequences are screened and if desiredused for further shuffling cycles. Synthetic shuffling techniques aredisclosed in WO 00/42561. In silico shuffling refers to a DNA shufflingprocedure which is performed or modelled using a computer system,thereby partly or entirely avoiding the need for physically manipulatingnucleic acids. Techniques for in silico shuffling are disclosed in WO00/42560.

[0182] Once assembled (by synthesis, site-directed mutagenesis oranother method), the nucleotide sequence encoding the polypeptide isinserted into a recombinant vector and operably linked to controlsequences necessary for expression of the G-CSF in the desiredtransformed host cell.

[0183] It should of course be understood that not all vectors andexpression control sequences function equally well to express thenucleotide sequence encoding a polypeptide described herein. Neitherwill all hosts function equally well with the same expression system.However, one of skill in the art may make a selection among thesevectors, expression control sequences and hosts without undueexperimentation. For example, in selecting a vector, the host must beconsidered because the vector must replicate in it or be able tointegrate into the chromosome. The vector's copy number, the ability tocontrol that copy number, and the expression of any other proteinsencoded by the vector, such as antibiotic markers, should also beconsidered. In selecting an expression control sequence, a variety offactors should also be considered. These include, for example, therelative strength of the sequence, its controllability, and itscompatibility with the nucleotide sequence encoding the polypeptide,particularly as regards potential secondary structures. Hosts should beselected by consideration of their compatibility with the chosen vector,the toxicity of the product coded for by the nucleotide sequence, theirsecretion characteristics, their ability to fold the polypeptidecorrectly, their fermentation or culture requirements, and the ease ofpurification of the products coded for by the nucleotide sequence.

[0184] The recombinant vector may be an autonomously replicating vector,i.e. a vector, which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector is one which, when introduced into ahost cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

[0185] The vector is preferably an expression vector in which thenucleotide sequence encoding the polypeptide of the invention isoperably linked to additional segments required for transcription of thenucleotide sequence. The vector is typically derived from plasmid orviral DNA. A number of suitable expression vectors for expression in thehost cells mentioned herein are commercially available or described inthe literature. Useful expression vectors for eukaryotic hosts include,for example, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Specific vectorsare, e.g., pcDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) andpCI-neo (Stratagene, La Jolla, Calif., USA). Useful expression vectorsfor yeast cells include the 2μ plasmid and derivatives thereof, the POT1vector (U.S. Pat. No. 4,931,373), the pJSO37 vector described in Okkels,Ann. New York Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C(Invitrogen). Useful vectors for insect cells include pVL941, pBG311(Cate et al., “Isolation of the Bovine and Human Genes for MullerianInhibiting Substance And Expression of the Human Gene In Animal Cells”,Cell, 45, pp. 685-98 (1986), pBluebac 4.5 and pMelbac (both availablefrom Invitrogen). Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, includingpBR322, pET3a and pET12a (both from Novagen Inc., WI, USA), wider hostrange plasmids, such as RP4, phage DNAs, e.g. the numerous derivativesof phage lambda, e.g. NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages.

[0186] Other vectors for use in this invention include those that allowthe nucleotide sequence encoding the polypeptide to be amplified in copynumber. Such amplifiable vectors are well known in the art. Theyinclude, for example, vectors able to be amplified by DHFR amplification(see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp,“Construction Of A Modular Dihydrafolate Reductase cDNA Gene: AnalysisOf Signals Utilized For Efficient Expression”, Mol. Cell. Biol., 2, pp.1304-19 (1982)) and glutamine synthetase (“GS”) amplification (see,e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

[0187] The recombinant vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV40 origin of replication. When the host cell is a yeast cell,suitable sequences enabling the vector to replicate are the yeastplasmid 2μ replication genes REP 1-3 and origin of replication.

[0188] The vector may also comprise a selectable marker, e.g. a genewhose product complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g. ampicillin, kanamycin,tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. ForSaccharomyces cerevisiae, selectable markers include ura3 and leu2. Forfilamentous fungi, selectable markers include amdS, pyrG, arcB, niaD andsC.

[0189] The term “control sequences” is defined herein to include allcomponents which are necessary or advantageous for the expression of thepolypeptide of the invention. Each control sequence may be native orforeign to the nucleic acid sequence encoding the polypeptide. Suchcontrol sequences include, but are not limited to, a leader sequence,polyadenylation sequence, propeptide sequence, promoter, enhancer orupstream activating sequence, signal peptide sequence, and transcriptionterminator. At a minimum, the control sequences include a promoter.

[0190] A wide variety of expression control sequences may be used in thepresent invention. Such useful expression control sequences include theexpression control sequences associated with structural genes of theforegoing expression vectors as well as any sequence known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof.

[0191] Examples of suitable control sequences for directingtranscription in mammalian cells include the early and late promoters ofSV40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1(metallothionein gene) promoter, the human cytomegalovirusimmediate-early gene promoter (CMV), the human elongation factor 1α(EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter,the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)promoter, the human growth hormone terminator, SV40 or adenovirus E1region polyadenylation signals and the Kozak consensus sequence (Kozak,M. J Mol Biol 1987 Aug 20;196(4):947-50).

[0192] In order to improve expression in mammalian cells a syntheticintron may be inserted in the 5′ untranslated region of the nucleotidesequence encoding the polypeptide. An example of a synthetic intron isthe synthetic intron from the plasmid pCI-Neo (available from PromegaCorporation, WI, USA).

[0193] Examples of suitable control sequences for directingtranscription in insect cells include the polyhedrin promoter, the P10promoter, the Autographa californica polyhedrosis virus basic proteinpromoter, the baculovirus immediate early gene 1 promoter, thebaculovirus 39K delayed-early gene promoter, and the SV40polyadenylation sequence. Examples of suitable control sequences for usein yeast host cells include the promoters of the yeast α-mating system,the yeast triose phosphate isomerase (TPI) promoter, promoters fromyeast glycolytic genes or alcohol dehydrogenase genes, the ADH2-4cpromoter, and the inducible GAL promoter. Examples of suitable controlsequences for use in filamentous fungal host cells include the ADH3promoter and terminator, a promoter derived from the genes encodingAspergillus oryzae TAKA amylase triose phosphate isomerase or alkalineprotease, an A. niger α-amylase, A. niger or A. nidulans glucoamylase,A. nidulans acetamidase, Rhizomucor miehei aspartic proteinase orlipase, the TPI1 terminator and the ADH3 terminator. Examples ofsuitable control sequences for use in bacterial host cells includepromoters of the lac system, the trp system, the TAC or TRC system, andthe major promoter regions of phage lambda.

[0194] The presence or absence of a signal peptide will, e.g., depend onthe expression host cell used for the production of the polypeptide tobe expressed (whether it is an intracellular or extracellularpolypeptide) and whether it is desirable to obtain secretion. For use infilamentous fungi, the signal peptide may conveniently be derived from agene encoding an Aspergillus sp. amylase or glucoamylase, a geneencoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosalipase. The signal peptide is preferably derived from a gene encoding A.oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid-stableamylase, or A. niger glucoamylase. For use in insect cells, the signalpeptide may conveniently be derived from an insect gene (cf. WO90/05783), such as the Lepidopteran manduca sexta adipokinetic hormoneprecursor, (cf. U.S. Pat. No. 5,023,328), the honeybee melittin(Invitrogen), ecdysteroid UDPglucosyltransferase (egt) (Murphy et al.,Protein Expression and Purification 4, 349-357 (1993) or humanpancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997).A preferred signal peptide for use in mammalian cells is that of hG-CSFor the murine Ig kappa light chain signal peptide (Coloma, M (1992) J.Imm. Methods 152:89-104). For use in yeast cells suitable signalpeptides have been found to be the α-factor signal peptide from S.cereviciae (cf. U.S. Pat. No. 4,870,008), a modified carboxypeptidasesignal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887-897), theyeast BAR1 signal peptide (cf. WO 87/02670), the yeast aspartic protease3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp.127-137), and the synthetic leader sequence TA57 (WO98/32867). For usein E. coli cells a suitable signal peptide has been found to be thesignal peptide ompA.

[0195] The nucleotide sequence of the invention encoding a polypeptideexhibiting G-CSF activity, whether prepared by site-directedmutagenesis, synthesis, PCR or other methods, may optionally alsoinclude a nucleotide sequence that encodes a signal peptide. The signalpeptide is present when the polypeptide is to be secreted from the cellsin which it is expressed. Such signal peptide, if present, should be onerecognized by the cell chosen for expression of the polypeptide. Thesignal peptide may be homologous (e.g. be that normally associated withhG-CSF) or heterologous (i.e. originating from another source thanhG-CSF) to the polypeptide or may be homologous or heterologous to thehost cell, i.e. be a signal peptide normally expressed from the hostcell or one which is not normally expressed from the host cell.Accordingly, the signal peptide may be prokaryotic, e.g. derived from abacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian,or insect or yeast cell.

[0196] Any suitable host may be used to produce the polypeptide orpolypeptide part of the conjugate of the invention, including bacteria,fungi (including yeasts), plant, insect, mammal, or other appropriateanimal cells or cell lines, as well as transgenic animals or plants.Examples of bacterial host cells include gram-positive bacteria such asstrains of Bacillus, e.g. B. brevis or B. subtilis, or Streptomyces, orgram-negative bacteria, such as strains of E. coli or Pseudomonas. Theintroduction of a vector into a bacterial host cell may, for instance,be effected by protoplast transformation (see, e.g., Chang and Cohen,1979, Molecular General Genetics 168: 111-115), using competent cells(see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThome, 1987, Journal of Bacteriology 169: 5771-5278). Examples ofsuitable filamentous fungal host cells include strains of Aspergillus,e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma.Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andU.S. Pat. No. 5,679,543. Suitable methods for transforming Fusariumspecies are described by Malardier et al., 1989, Gene 78: 147-156 and WO96/00787. Examples of suitable yeast host cells include strains ofSaccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Klyveromyces,Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H.polymorpha or Yarrowia. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920: and asdisclosed by Clontech Laboratories, Inc, Palo Alto, Calif., USA (in theproduct protocol for the Yeastmaker™ Yeast Transformation System Kit).Examples of suitable insect host cells include a Lepidoptora cell line,such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells(High Five) (U.S. Pat. No. 5,077,214). Transformation of insect cellsand production of heterologous polypeptides therein may be performed asdescribed by Invitrogen. Examples of suitable mammalian host cellsinclude Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCCCCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK)cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g.HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Rockville, Md. Methods for introducing exogeneous DNA into mammalianhost cells include calcium phosphate-mediated transfection,electroporation, DEAE-dextran mediated transfection, liposome-mediatedtransfection, viral vectors and the transfection method described byLife Technologies Ltd, Paisley, UK using Lipofectamin 2000. Thesemethods are well known in the art and e.g. described by Ausbel et al.(eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons,New York, USA. The cultivation of mammalian cells is conducted accordingto established methods, e.g. as disclosed in (Animal Cell Biotechnology,Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc,Totowa, N.J., USA and Harrison M A and Rae I F, General Techniques ofCell Culture, Cambridge University Press 1997).

[0197] In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermenters performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

[0198] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray drying, evaporation, orprecipitation.

[0199] The polypeptides may be purified by a variety of procedures knownin the art including, but not limited to, chromatography (e.g., ionexchange, affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989). Specificmethods for purifying polypeptides exhibiting G-CSF activity aredescribed by D. Metcalf and N. A. Nicola in The hemopoieticcolony-stimulating factors, p. 50-51, Cambridge University Press (1995),by C. S. Bae et al., Appl. Microbiol. Biotechnol, 52:338-344 (1999) andin U.S. Pat. No. 4,810,643.

[0200] Pharmaceutical Composition of the Invention and Its Use

[0201] In a further aspect, the present invention comprises acomposition comprising a polypeptide or conjugate as described hereinand at least one pharmaceutically acceptable carrier or excipient.

[0202] The polypeptide, the conjugate or the pharmaceutical compositionaccording to the invention may be used for the manufacture of amedicament for treatment of diseases, in particular prevention ofinfection in cancer patients undergoing certain types of chemotherapy,radiation therapy and bone marrow transplantations, mobilisation ofprogenitor cells for collection in peripheral blood progenitor celltransplantations, treatment of severe chronic or relative leukopenia,treatment of patients with acute mycloid leukaemia, treatment of AIDS orother immunodeficiency diseases, and for antifungal therapy, inparticular for treatment of systemic or invasive candidiasis.

[0203] In another aspect the polypeptide, the conjugate or thepharmaceutical composition according to the invention is used in amethod of treating a mammal having a general haematopoietic disorder,including those arising from radiation therapy or from chemotherapy, inparticular neutropenia or leukopenia, AIDS or other immunodeficiencydiseases, comprising administering to a mammal in need thereof such apolypeptide, conjugate or pharmaceutical composition. In particular, themethod is aimed at increasing the level of neutrophils in a patientsuffering from an insufficient neutrophil level, for example due tochemotherapy, radiation therapy, or HIV or another viral infection.

[0204] The polypeptides and conjugates of the invention will beadministered to patients in a “therapeutically effective” dose, i.e. adose that is sufficient to produced the desired effects in relation tothe condition for which it is administered. The exact dose will dependon the disorder to be treated, and will be ascertainable by one skilledin the art using known techniques. The polypeptides or conjugates of theinvention may e.g. be administered at a dose similar to that employed intherapy with rhG-CSF such as Neupogen®. A suitable dose of a conjugateof the invention is contemplated to be in the range of about 5-300microgram/kg body weight (based on the weight of the protein part of theconjugate), e.g. 10-200 microgram/kg, such as 25-100 microgram/kg. Itwill be apparent to those of skill in the art that an effective amountof a polypeptide, conjugate or composition of the invention depends,inter alia, upon the disease, the dose, the administration schedule,whether the polypeptide or conjugate or composition is administeredalone or in conjunction with other therapeutic agents, the serumhalf-life of the compositions, the general health of the patient, andthe frequency of administration. Preferably, the polypeptide, conjugate,preparation or composition of the invention is administered in aneffective dose, in particular a dose which is sufficient to normalizethe number of leukocytes, in particular neutrophils, in the patient inquestion. Normalization of the number of leukocytes may be determined bysimply counting the number of leukocytes at regular intervals inaccordance with established practice.

[0205] The polypeptide or conjugate of the invention is preferablyadministered in a composition including one or more pharmaceuticallyacceptable carriers or excipients. The polypeptide or conjugate can beformulated into pharmaceutical compositions in a manner known per se inthe art to result in a polypeptide pharmaceutical that is sufficientlystorage-stable and is suitable for administration to humans or animals.The pharmaceutical composition may be formulated in a variety of forms,including as a liquid or gel, or lyophilized, or any other suitableform. The preferred form will depend upon the particular indicationbeing treated and will be apparent to one of skill in the art.

[0206] Accordingly, this invention provides compositions and methods fortreating various forms of leukopenia or neutropenia. In particular thepolypeptide, conjugate or composition of the invention may be used toprevent infection in cancer patients undergoing certain types ofradiation therapy chemotherapy and bone marrow transplantations, tomobilize progenitor cells for collection in peripheral blood progenitorcell transplantations, for treatment of severe chronic or relativeleukopenia and to support treatment of patients with acute myeloidleukaemia. Additionally, the polypeptide, conjugate or composition ofthe invention may be used for treatment of AIDS or otherimmunodeficiency diseases and for antifungal therapy, in particular fortreament of systemic or invasive candidiasis, and for the treatment ofbacterial infections.

[0207] Since the polypeptide conjugates of the invention have a long invivo half-life and have been found to reduce the duration of neutropeniaand leukopenia by administration of a single dose, in contrast to hG-CSFwhich must be administered daily, the conjugates of the invention arewell-suited for administration e.g. on a weekly basis for the preventionand/or treatment of neutropenia. In one embodiment, the polypeptideconjugate or pharmaceutical composition of the invention is for theprevention and/or treatment of neutropenia due to chemotherapy. In thecase of chemotherapy administered at intervals, e.g. on a weekly basisby intravenous injection or by another type of injection, such assubcutaneous or intramuscular injection, it will normally be sufficientto administer the conjugate of the invention in a single dose perchemotherapy treatment, i.e. given either before, after orsimultaneously with the chemotherapy. In other cases where thechemotherapy is administered differently, for example orally on a dailybasis or over an extended period of time by means of an infusion pump,the conjugates of the invention may be administered in a similar manner,e.g. once a week, or, in the case of chemotherapy sessions given lessfrequently than once a week, once per session.

[0208] Drug Form

[0209] The polypeptide or conjugate of the invention can be used “as is”and/or in a salt form thereof. Suitable salts include, but are notlimited to, salts with alkali metals or alkaline earth metals, such assodium, potassium, calcium and magnesium, as well as e.g. zinc salts.These salts or complexes may by present as a crystalline and/oramorphous structure.

[0210] Excipients

[0211] “Pharmaceutically acceptable” means a carrier or excipient thatat the dosages and concentrations employed does not cause any untowardeffects in the patients to whom it is administered. Suchpharmaceutically acceptable carriers and excipients are well known inthe art (see Remington's Pharmaceutical Sciences, 18th edition, A. R.Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical FormulationDevelopment of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds.,Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rdedition, A. Kibbe, Ed., Pharmaceutical Press [2000]).

[0212] Mix of Drugs

[0213] The pharmaceutical composition of the invention may beadministered alone or in conjunction with other therapeutic agents.These agents may be incorporated as part of the same pharmaceuticalcomposition or may be administered separately from the polypeptide orconjugate of the invention, either concurrently or in accordance withanother treatment schedule. In addition, the polypeptide, conjugate orpharmaceutical composition of the invention may be used as an adjuvantto other therapies.

[0214] Patients

[0215] A “patient” for the purposes of the present invention includesboth humans and other mammals. Thus the methods are applicable to bothhuman therapy and veterinary applications.

[0216] Administration Route

[0217] The administration of the formulations of the present inventioncan be performed in a variety of ways, including, but not limited to,orally, subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner.The formulations can be administered continuously by infusion, althoughbolus injection is acceptable, using techniques well known in the art.Typically, the formulation will designed for parenteral administration,e.g. by the subcutaneous route.

[0218] Parenterals

[0219] An example of a pharmaceutical composition is a solution designedfor parenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

[0220] In case of parenterals, they are prepared for storage aslyophilized formulations or aqueous solutions by mixing, as appropriate,the polypeptide having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and/or othermiscellaneous additives.

[0221] Buffering agents help to maintain the pH in the range whichapproximates physiological conditions. They are typically present at aconcentration ranging from about 2 mM to about 50 mM Suitable bufferingagents for use with the present invention include both organic andinorganic acids and salts thereof such as citrate buffers (e.g.,monosodium citrate-disodium citrate mixture, citric acid-trisodiumcitrate mixture, citric acid-monosodium citrate mixture, etc.),succinate buffers (e.g., succinic acid-monosodium succinate mixture,succinic acid-sodium hydroxide mixture, succinic acid-disodium succinatemixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartratemixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodiumhydroxide mixture, etc.), fumarate buffers (e.g., fumaricacid-monosodium fumarate mixture, fumaric acid-di sodium fumaratemixture, monosodium fumarate-di sodium fumarate mixture, etc.),gluconate buffers (e.g., gluconic acid-sodium glyconate mixture,gluconic acid-sodium hydroxide mixture, gluconic acid-potassiumglyuconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodiumoxalate mixture, oxalic acid-sodium hydroxide mixture, oxalicacid-potassium oxalate mixture, etc.), lactate buffers (e.g., lacticacid-sodium lactate mixture, lactic acid-sodium hydroxide mixture,lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g.,acetic acid-sodium acetate mixture, acetic acid-sodium hydroxidemixture, etc.). Additional possibilities are phosphate buffers,histidine buffers and trimethylamine salts such as Tris.

[0222] Preservatives are added to retard microbial growth, and aretypically added in amounts of about 0.2%-1% (w/v). Suitablepreservatives for use with the present invention include phenol, benzylalcohol, meta-cresol, methyl paraben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g.benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkylparabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol and 3-pentanol.

[0223] Isotonicifiers are added to ensure isotonicity of liquidcompositions and include polyhydric sugar alcohols, preferably trihydricor higher sugar alcohols, such as glycerin, erythritol, arabitol,xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in anamount between 0.1% and 25% by weight, typically 1% to 5%, taking intoaccount the relative amounts of the other ingredients.

[0224] Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

[0225] Non-ionic surfactants or detergents (also known as “wettingagents”) may be present to help solubilize the therapeutic agent as wellas to protect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.).

[0226] Additional miscellaneous excipients include bulking agents orfillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

[0227] The active ingredient may also be entrapped in microcapsulesprepared, for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

[0228] Parenteral formulations to be used for in vivo administrationmust be sterile. This is readily accomplished, for example, byfiltration through sterile filtration membranes.

[0229] Sustained Release Preparations

[0230] Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thepolypeptide or conjugate, the matrices having a suitable form such as afilm or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0231] All references cited herein are hereby incorporated by referencein their entirety for all purposes.

[0232] The invention is further described in the non-limiting examplesbelow.

DESCRIPTION OF THE DRAWINGS

[0233]FIG. 1: The in vivo half-lives of rhG-CSF (Neupogen®) and SPA-PEG5000-conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K

[0234]FIG. 2: The in vivo half-lives of rhG-CSF (Neupogen®) and SPA-PEG5000-conjugated hG-CSF K16R K34R K40R Q90K T105K Q159K

[0235]FIG. 3: The in vivo biological activities in healthy rats ofrhG-CSF (Neupogen®), SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70KQ120K and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K.

[0236]FIG. 4: The in vivo biological activities in healthy rats ofrhG-CSF (Neupogen®), SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70KQ120K T133K and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q90K Q120KT133K.

[0237]FIG. 5: The in vivo biological activities in healthy rats ofrhG-CSF (Neupogen®), SPA-PEG 12000-conjugated hG-CSF K16R K34R K40R anddifferent doses of SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70KQ90K Q120K.

[0238]FIG. 6: The in vivo biological activities in healthy rats ofrhG-CSF (Neupogen®), SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70KQ90K Q120K, SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q90K T105KS159K and SPA-PEG 20000-conjugated hG-CSF K16R K34R K40R T105K S159K.

[0239]FIG. 7: The in vivo biological activities in rats withchemotherapy-induced neutropenia of rhG-CSF (Neupogen®), SPA-PEG5000-conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K, SPA-PEG20000-conjugated hG-CSF K16R K34R K40R Q90K.

[0240]FIG. 8: The in vivo biological activities (white blood cell count)in rats with chemotherapy-induced neutropenia of rhG-CSF (Neupogen®),SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R T105K S159K, and SPA-PEG5000-conjugated hG-CSF K16R K34R K40R Q90K T105K S159K.

[0241]FIG. 9: The in vivo biological activities (absolute neutrophilcount) in rats with chemotherapy-induced neutropenia of rhG-CSF(Neupogen®) and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R T105KS159K.

[0242]FIG. 10: The in vivo biological activities (white blood cellcount) in rats with chemotherapy-induced neutropenia of rhG-CSF(Neupogen®), rhG-CSF with a 20 kDa N-terminal PEG group (Neulasta™), andSPA-PEG 5000-conjugated hG-CSF K16R K34R K40R T105K S159K produced inyeast and in CHO cells.

[0243]FIG. 11: The in vivo biological activities (absolute neutrophilcount) in rats with chemotherapy-induced neutropenia of rhG-CSF(Neupogen®), rhG-CSF with a 20 kDa N-terminal PEG group (Neulasta™), andSPA-PEG 5000-conjugated hG-CSF K16R K34R K40R T105K S159K produced inyeast and in CHO cells.

SEQUENCE LISTING

[0244] The appended sequence listing contains the following sequences:

[0245] SEQ ID NO: 1: The amino acid sequence of human G-CSF.

[0246] SEQ ID NO:2: A synthetic DNA sequence encoding human G-CSF, withcodon usage optimized for expression in E. coli.

[0247] SEQ ID NO:3: The amino acid sequence of the OmpA signal sequence.

[0248] SEQ ID NO:4: A synthetic DNA sequence encoding the OmpA signalsequence.

[0249] SEQ ID NO:5: A synthetic histidine tag.

[0250] SEQ ID NO:6: A synthetic DNA sequence encoding the histidine tagof SEQ ID NO:5.

[0251] SEQ ID NO:7: The amino acid sequence of a human G-CSF signalpeptide.

[0252] SEQ ID NO:8: A synthetic DNA sequence encoding human G-CSF,including the signal peptide of SEQ ID NO:7, with codon usage optimizedfor expression in CHO cells.

[0253] SEQ ID NO:9-15: Various synthetic tags

MATERIALS AND METHODS

[0254] Methods Used to Determine the Amino Acids to be Modified

[0255] Accessible Surface Area (ASA)

[0256] A 3D ensemble of 10 structures determined by NMR spectroscopy(Zink et al. (1994) Biochemistry 33: 8453-8463) is available from theProtein Data Bank (PDB) (www.rcsb.org/pdb/). This information can beentered into the computer program Access (B. Lee and F. M. Richards, J.Mol. Biol. 55: 379-400 (1971)) version 2 ((© 1983 Yale University) andused to compute the accessible surface area (ASA) of the individualatoms in the structure. This method typically uses a probe size of 1.4Aand defines the Accessible Surface Area (ASA) as the area formed by thecentre of the probe. Prior to this calculation all water molecules andall hydrogen atoms should be removed from the coordinate set as shouldother atoms not directly related to the protein.

[0257] Fractional ASA of Side Chain

[0258] The fractional ASA of the side chain atoms is computed bydivision of the sum of the ASA of the atoms in the side chain with avalue representing the ASA of the side chain atoms of that residue typein an extended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton(1991) J. Mol. Biol. 220, 507-530. For this example the CA atom isregarded as a part of the side chain of glycine residues but not for theremaining residues. The values in the following table are used asstandard 100% ASA for the side chain: Ala 69.23 Å² Leu 140.76 Å² Arg200.35 Å² Lys 162.50 Å² Asn 106.25 Å² Met 156.08 Å² Asp 102.06 Å² Phe163.90 Å² Cys 96.69 Å² Pro 119.65 Å² Gln 140.58 Å² Ser 78.16 Å² Glu134.61 Å² Thr 101.67 Å² Gly 32.28 Å² Trp 210.89 Å² His 147.00 Å² Tyr176.61 Å² Ile 137.91 Å² Val 114.14 Å²

[0259] Residues not detected in the structure are defined as having 100%exposure as they are thought to reside in flexible regions.

[0260] Determining Distances Between Atoms:

[0261] The distance between atoms is most easily determined usingmolecular graphics software, e.g. InsightII® v. 98.0, MSI INC.

[0262] General Considerations Regarding Amino Acid Residues to beModified

[0263] As explained above, amino acid residues to be modified inaccordance with the present invention are preferably those whose sidechains are surface exposed, in particular those with more than about 25%of the side chain exposed at the surface of the molecule, and morepreferably those with more than 50% side chain exposure. Anotherconsideration is that residues located in receptor interfaces arepreferably excluded so as to avoid or at least minimize possibleinterference with receptor binding or activation. A furtherconsideration is that residues that are less than 10 Å from the nearestLys (Glu, Asp) CB-CB (CA for Gly) should also be excluded. Finally,preferred positions for modification are in particular those that have ahydrophilic and/or charged residue, i.e. Asp, Asn, Glu, Gln, Arg, His,Tyr, Ser and Thr, positions that have an arginine residue beingespecially preferred.

[0264] Identifying G-CSF Amino Acid Residues for Modification

[0265] The information below illustrates the factors that generallyshould be taken into consideration when identifying amino acid residuesto be modified in accordance with the present invention.

[0266] Three-dimensional structures have been reported for human G-CSFby X-ray crystallography (Hill et al. (1993) Proc. Natl. Acad. Sci. USA90: 5167-5171) and by NMR spectroscopy (Zink et al. (1994) Biochemistry33: 8453-8463). As mentioned above, Aritomi et al. (Nature 401:713-717,1999) have identified the following hG-CSF residues as being part of thereceptor binding interfaces: G4, P5, A6, S7, S8, L9, P10, Q11, S12, L15,K16, E19, Q20, o L108, D109, D112, T115, T116, Q119, E122, E123, andL124. Thus, although it is possible to modify these residues, it ispreferred that these residues are excluded from modification.

[0267] Using the 10 NMR structures of G-CSF identified by Zink et al.(1994) as input structures followed by a computation of the average ASAof the side chain, the following residues have been identified as havingmore than 25% ASA: M0, T1, P2, L3, G4, P5, A6, S7, S8, L9, P10, Q11,S12, F13, L14, L15, K16, C17, E19, Q20, V21, R22, K23, Q25, G26, D27,A29, A30, E33, K34, C36, A37, T38, Y39, K40, L41, H43, P44, E45, E46,V48, L49, L50, H52, S53, L54, 156, P57, P60, L61, S62, S63, P65, S66,Q67, A68, L69, Q70, L71, A72, G73, C74, S76, Q77, L78, S80, F83, Q86,G87, Q90, E93, G94, S96, P97, E98, L99, G100, P101, T102, D104, T105,Q107, L108, D109, A111, D112, F113, T115, T116, W118, Q119, Q120, M121,E122, E123, L124, M126, A127, P128, A129, L130, Q131, P132, T133, Q134,G135, A136, M137, P138, A139, A141, S142, A143, F144, Q145, R146, R147,S155, H156, Q158, S159, L161, E162, V163, S164, Y165, R166, V167, L168,R169, H170, L171, A172, Q173, P174.

[0268] Similarly, the following residues have more than 50% ASA: M0, T1,P2, L3, G4, P5, A6, S7, S8, L9, P10, Q11, S12, F13, L14, L15, K16, C17,E19, Q20, R22, K23, G26, D27, A30, E33, K34, T38, K40, L41, H43, P44,E45, E46, L49, L50, S53, P57, P60, L61, S62, S63, P65, S66, Q67, A68,L69, Q70, L71, A72, G73, S80, F83, Q90, G94, P97, E98, P101, D104, T105,L108, D112, F113, T115, T116, Q119, Q120, E122, E123, L124, M126, P128,A129, L130, Q131, P132, T133, Q134, G135, A136, A139, A141, S142, A143,F144, R147, S155, S159, E162, R166, V167, R169, H170, L171, A172, Q173,P174.

[0269] The molecular graphics program InsightII® v.98.0 was used todetermine residues having their CB atom (CA in the case of glycine) at adistance of more than 15 Å from the nearest amine group, defined as theNZ atoms of lysine and the N atom of the N-terminal residue T1. Thefollowing list includes the residues that fulfill this criteria in atleast one of the 10 NMR structures. G4, P5, A6, S7, S8, L9, P10, Q11,L14, L15, L18, V21, R22, Q25, G26, D27, G28, A29, Q32, L35, C36, T38,Y39, C42, H43, P44, E45, E46, L47, V48, L49, L50, G51, H52, S53, L54,G55, 156, P57, W58, A59, P60, L61, S62, S63, C64, P65, S66, Q67, A68,L69, Q70, L71, A72, G73, C74, L75, S76, Q77, L78, H79, S80, G81, L82,F83, L84, Y85, Q86, G87, L88, L89, Q90, A91, L92, E93, G94, 195, S96,P97, E98, L99, G100, P101, T102, L103, D104, T105, L106, Q107, L108,D109, V110, A111, D112, F113, A114, T115, T116, I117, W118, Q119, Q120,M121, E122, E123, L124, G125, M126, A127, P128, A129, L130, Q131, P132,T133, Q134, G135, A136, M137, P138, A139, F140, A141, S142, A143, F144,Q145, R146, R147, A148, G149, G150, V151, L152, V153, A154, S155, H156,L157, Q158, S159, F160, L161, E162, V163, S164, Y165, R166, V167, L168,R169, H170, L171, A172, Q173, P174.

[0270] The InsightII® v.98.0 program was similarly used to determineresidues having their CB atom (CA atom in the case of glycine) at adistance of more than 10 Å from the nearest acidic group, defined as theCG atoms of aspartic acid, the CD atoms of glutamic acid and the C atomof the C-terminal residue P174. The following list includes the residuesthat fulfill this criteria in at least one of the 10 NMR structures. M0,T1, P2, L3, G4, P5, A6, S7, S8, L9, P10, Q11, S12, F13, L14, T38, Y39,K40, L41, C42, L50, G51, H52, S53, L54, G55, 156, P57, W58, A59, P60,L61, S62, S63, C64, P65, S66, Q67, A68, L69, Q70, L71, A72, G73, C74,L75, S76, Q77, L78, H79, S80, G81, L82, F83, L84, Y85, Q86, G87, L88,1117, M126, A127, P128, A129, L130, Q131, P132, T133, Q134, G135, A136,M137, P138, A139, F140, A141, S142, A143, F144, Q145, R146, R147, A148,G149, G150, V151, L152, V153, A154, S155, H156, L157, V167, L168, R169,H170, L171.

[0271] By combining and comparing the above lists, it is possible toselect individual amino acid residues for modification to result in alist containing a limited number of amino acid residues whosemodification in a given G-CSF polypeptide is likely to result in desiredproperties.

[0272] Methods for PEGylation of hG-CSF and Variants Thereof

[0273] PEGylation of hG-CSF and Variants Thereof in Solution

[0274] Human G-CSF and variants thereof are PEGylated at a concentrationof 250 μg/ml in 50 mM sodium phosphate, 100 mM NaCl, pH 8.5. The molarsurplus of PEG is 50-100 times with respect to PEGylation sites on theprotein. The reaction mixture is placed in a thermo mixer for 30 minutesat 37° C. at 1200 rpm. After 30 minutes, quenching of the reaction isobtained by adding a molar excess of glycine.

[0275] Cation exchange chromatography is applied to remove excess PEG,glycine and other by-products from the reaction mixture. The PEGylationreaction mixture is diluted with 20 mM sodium citrate pH 2.5 until theionic strength is less than 7 mS/cm. pH is adjusted to 2.5 using 5 NHCl. The mixture is applied to a SP-sepharose FF column equilibratedwith 20 mM sodium citrate pH 2.5. Unbound material is washed off thecolumn using 4 column volumes of equilibration buffer. PEGylated proteinis eluted in three column volumes by adding 20 mM sodium citrate, 750 mMsodium chloride. Pure PEGylated G-CSF is concentrated and bufferexchange is performed using VivaSpin concentration devices, molecularweight cut-off (mwco): 10 kDa.

[0276] PEGylation in Microtiter Plates of a Tagged Polypeptide withG-CSF Activity

[0277] A polypeptide exhibiting G-CSF activity is expressed with asuitable tag, e.g. any of the tags exemplified in the generaldescription above, and culture broth is transferred to one or more wellsof a microtiter plate capable of immobilising the tagged polypeptide.When the tag isMet-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-Gln (SEQ IDNO:5), a nickel-nitrilotriacetic acid (Ni-NTA) H is Sorb microtiterplate commercially available from QIAGEN can be used.

[0278] After immobilization of the tagged polypeptide to the microtiterplate, the wells are washed in a buffer suitable for binding andsubsequent PEGylation followed by incubating the wells with theactivated PEG of choice. As an example, M-SPA-5000 from Shearwater Corp.is used. The molar ratio of activated PEG to polypeptide should beoptimized, but will typically be greater than 10: 1, e.g. up to about100:1 or higher. After a suitable reaction time at ambient temperature,typically around 1 hour, the reaction is stopped by removal of theactivated PEG solution. The conjugated protein is eluted from the plateby incubation with a suitable buffer. Suitable elution buffers maycontain imidazole, excess NTA or another chelating compound. Theconjugated protein is assayed for biological activity and immunogenicityas appropriate. The tag may optionally be cleaved off using a methodknown in the art, e.g. using diaminopeptidase the Gln in pos −1 can beconverted to pyroglutamyl with GCT (glutamylcyclotransferase) andfinally cleaved off with PGAP (pyro-glutamyl-aminopeptidase), giving theuntagged protein. The process involves several steps of metal chelateaffinity chromatography. Alternatively, the tagged polypeptide may beconjugated.

[0279] PEGylation of a Polypeptide Exhibiting hG-CSF Activity and Havinga Blocked Receptor-Binding Site

[0280] In order to optimize PEGylation of hG-CSF in a manner excludingPEGylation of lysines involved in receptor recognition, the followingmethod has been developed: Purified hG-CSF is obtained as described inExample 4. A homodimer complex consisting of an hG-CSF polypeptide andthe soluble domain of the G-CSF receptor in a 2:2 stochiometry is formedin a phosphate-buffered saline solution (PBS) buffer at pH 7. Theconcentration of hG-CSF polypeptide is approximately 20 μg/ml or 1 μMand the receptor is present at an equimolar concentration.

[0281] M-SPA-5000 from Shearwater Corp. is added at 3 differentconcentration levels corresponding to a 10, 25 and 50 molar excess tothe number of attachment sites in hG-CSF polypeptide. The reaction timeis 30 min at room temperature. After the 30 min reaction period, the pHof the reaction mixture is adjusted to pH 2.0 and the reaction mixtureis applied to a Vydac C18 column and eluted with an acetonitrilegradient essentially as described (Utsumi et al., J. Biochem., Vol. 101,1199-1208, (1987). Alternatively, an isopropanol gradient can be used.

[0282] Fractions are analyzed using the primary screening assaydescribed herein and active PEGylated hG-CSF polypeptide obtained bythis method is stored at −80° C. in PBS, pH 7 containing 1 mg/ml humanserum albumin (HSA).

[0283] Methods Used to Characterize Conjugated and Non-Conjugated hG-CSFand Variants Thereof

[0284] Determination of the Molecular Size of hG-CSF and VariantsThereof

[0285] The molecular weight of conjugated or non-conjugated hG-CSF orvariants thereof is determined by either SDS-PAGE, gel filtration,matrix assisted laser desorption mass spectrometry or equilibriumcentrifugation. As explained above, SDS-PAGE provides information on the“apparent molecular weight”. The actual molecular weight canadvantageously be determined using mass spectrometry. SDS-PAGE iscarried out using the NuPAGE® kit (Novex high-performance pre-cast gels)from Invitrogen™. 15 μt of the samples are loaded onto NuPAGE 4-12%Bis-Tris gels (Cat. Nr. NPO₃₂₁) and eluted in NuPAGE MES SDS runningbuffer (Cat. Nr. NPO002-₀₂) for 35 minutes at 200 V and 120 mA.

[0286] Determination of Polypeptide Concentration

[0287] The concentration of a polypeptide can be measured using opticaldensity measurements at 280 nm, an enzyme-linked immunoadsorption assay(ELISA), a radio-immunoassay (RIA), or other such immunodetectiontechniques well known in the art. Furthermore, the polypeptideconcentration in a sample can be measured with the Biacore® instrumentusing a Biacore® chip coated with an antibody specific for thepolypeptide.

[0288] Such an antibody can be coupled covalently to the Biacore®(! chipby various chemistries. Alternatively, the antibody can be boundnon-covalently e.g. by means of an antibody specific for the Fc portionof the anti-polypeptide antibody. The Fe specific antibody is firstcoupled covalently to the chip. The anti-polypeptide antibody is thenflowed over the chip and is bound by the first antibody in a directedfashion. Furthermore, biotinylated antibodies can be immobilized using astreptavidin coated surface (e.g. Biacore Sensor Chip SA®) (Real-TimeAnalysis of Biomolecular Interactions, Nagata and Handa (Eds.), 2000,Springer Verlag, Tokyo; Biacore 2000 Instrument Handbook, 1999, BiacoreAB).

[0289] When the sample is flowed over the chip the polypeptide will bindto the coated antibody and the increase in mass can be measured. Byusing a preparation of the polypeptide in a known concentration, astandard curve can be established and subsequently the concentration ofthe polypeptide in the sample can be determined. After each injection ofsample the sensor chip is regenerated by a suitable eluent (e.g. a lowpH buffer) that removes the bound analyte.

[0290] Generally, the applied antibodies will be monoclonal antibodiesraised against the wild type polypeptide. Introduction of mutations orother manipulations of the wild type polypeptide (extra glycosylationsor polymer conjugations) may alter the recognition by such antibodies.Furthermore, such manipulations that give rise to an increased molecularweight of the polypeptide will result in an increased plasmon resonancesignal. Consequently, it is necessary to establish a standard curve forevery molecule to be tested.

[0291] Methods Used to Determine the in vitro and in vivo Activity ofConjugated and Non-Conjugated hG-CSF and Variants Thereof

[0292] Primary Assay 1—in vitro G-CSF Activity Assay

[0293] Proliferation of the murine cell line NFS-60 (obtained from Dr.J. Ihle, St. Jude Children's Research Hospital, Tennessee, USA) isdependent on the presence of active G-SCF in the growth medium. Thus,the in vitro biological activity of hG-CSF and variants thereof can bedetermined by measuring the number of dividing NFS-60 cells afteraddition of a G-CSF sample to the growth medium followed by incubationover a fixed period of time.

[0294] NFS-60 cells are maintained in Iscoves DME Medium containing 10%w/w FBS (fetal bovine serum), 1% w/w Pen/Strep, 10 μg per liter hG-CSFand 2 mM Glutamax. Prior to sample addition, cells are washed twice ingrowth medium without hG-CSF and diluted to a concentration of 2.2×10⁵cells per ml. 100 μl of the cell suspension is added to each well of a96 well microtiter plate (Coming).

[0295] Samples containing conjugated or non-conjugated G-CSF or variantsthereof are diluted to concentrations between 1.1×10⁶ M and 1.1×10⁻¹³ Min the growth medium. 10 μt of each sample is added to 3 wellscontaining NFS-60 cells. A control consisting of 10 μl of mammaliangrowth medium is added to 8 wells on each microtiter plate. The cellsare incubated for 48 hours (37° C., 5% CO₂) and the number of dividingcells in each well is quantified using the WST-1 cell proliferationagent (Roche Diagnostics GmbH, Mannheim, Germany). 0.01 ml WST-1 isadded to the wells followed by incubation for 150 min. at 37° C. in a 5%CO₂ air atmosphere. The cleavage of the tetrazolium salt WST-1 bymitochondrial dehydrogenases in viable cells results in the formation offormazan that is quantified by measuring the absorbance at 450 nm.Hereby, the number of viable cells in each well is quantified.

[0296] Based on these measurements, dose-response curves for eachconjugated and non-conjugated G-CSF molecule or variants thereof arecalculated, after which the EC50 value for each molecule can bedetermined. This value is equal to the amount of active G-CSF proteinthat is necessary to obtain 50% of the maximum proliferation activity ofnon-conjugated human G-CSF. Thus, the EC50 value is a direct measurementof the in vitro activity of the given protein, a lower EC50 valueindicating a higher activity.

[0297] Primary Assay 2—in vitro G-CSF Activity Assay

[0298] The murine hematopoietic cell line BaF3 is transfected with aplasmid carrying the human G-CSF receptor and the promoter of thetranscription regulator, fos, in front of the luciferase reporter gene.Upon stimulation of such a cell line with a G-CSF sample, a number ofintracellular reactions lead to stimulation of fos expression, andconsequently to expression of luciferase. This stimulation is monitoredby the Steady-Glo™ Luciferase Assay System (Promega, Cat. No. E2510)whereby the in vitro activity of the G-CSF sample may be quantified.

[0299] BaF3/hGCSF—R/pfos-lux cells are maintained at 37° C. in ahumidified 5% CO₂ atmosphere in complete culture media (RPMI-1640/HEPES(Gibco/BRL, Cat. No. 22400), 10% FBS (HyClone, characterized), 1×Penicillin/Streptomycin (Gibco/BRL, Cat. No. 15140-122), 1× L-Glutamine(Gibco/BRL, Cat. No. 25030-081), 10% WEHI-3 conditioned media (source ofmuIL-3), and grown to a density of 5×10⁵ cells/mL (confluent). The cellsare reseeded at about 2×10⁴ cells/mL every 2-3 days.

[0300] One day prior to the assay, log-phase cells are resuspended at2×10⁵ cells/mL in starving media (DMEM/F-12 (Gibco/BRL, Cat. No. 11039),1% BSA (Sigma, Cat. No. A3675), 1× Penicillin/Streptomycin (Gibco/BRL,Cat. No. 15140-122), 1× L-Glutamine (Gibco/BRL, Cat. No. 25030-081),0.1% WEHI-3 conditioned media) and starved for 20 hours. The cells arewashed twice with PBS and tested for viability using Trypan Blueviability staining. The cells are resuspended in assay media (RPMI-1640(phenol-red free, Gibco/BRL, Cat. No. 11835), 25 mM HEPES, 1% BSA(Sigma, Cat. No. A3675), 1× Penicillin/Streptomycin (Gibco/BRL, Cat. No.15140-122), 1× L-Glutamine (Gibco/BRL, Cat. No. 25030-081) at 4×10⁶cells/mL, and 50 μL are aliquotted into each well of a 96-wellmicrotiter plate (Coming). Samples containing conjugated ornon-conjugated G-CSF or variants thereof are diluted to concentrationsbetween 1.1×10⁻⁷ M and 1.1×10⁻¹² M in the assay medium. 50 μl of eachsample is added to 3 wells containing BaF3/hGCSF—R/pfos-lux cells. Anegative control consisting of 50 μl of medium is added to 8 wells oneach microtiter plate. The plates are mixed gently and incubated for 2hours at 37° C. The luciferase activity is measured by following thePromega Steady-Glo™ protocol (Promega Steady-Glo™ Luciferase AssaySystem, Cat. No. E2510). 100 μL of substrate is added per well followedby gentle mixing. Luminescence is measured on a TopCount luminometer(Packard) in SPC (single photon counting) mode.

[0301] Based on these measurements, dose-response curves for eachconjugated and non-conjugated G-CSF molecule or variants thereof arecalculated, after which the EC50 value for each molecule can bedetermined.

[0302] Secondary Assay—Binding Affinity of G-CSF or Variants Thereof tothe hG-CSF Receptor

[0303] Binding of rhG-CSF or variants thereof to the hG-CSF receptor isstudied using standard binding assays. The receptors may be purifiedextracellular receptor domains, receptors bound to purified cellularplasma membranes, or whole cells—the cellular sources being either celllines that inherently express G-CSF receptors (e.g. NFS-60) or cellstransfected with cDNAs encoding the receptors. The ability of rhG-CSF orvariants thereof to compete for the binding sites with native G-CSF isanalyzed by incubating with a labeled G-CSF-analog, for instancebiotinylated hG-CSF or radioiodinated hG-CSF. An example of such anassay is described by Yamasaki et al. (Drugs. Exptl. Clin. Res.24:191-196 (1998)).

[0304] The extracellular domains of the hG-CSF receptor can optionallybe coupled to Fe and immobilized in 96 well plates. RhG-CSF or variantsthereof are subsequently added and the binding of these is detectedusing either specific anti-hG-CSF antibodies or biotinylated orradioiodinated hG-CSF.

[0305] Measurement of the in vivo Half-Life of Conjugated andNon-Conjugated rhG-CSF and Variants Thereof

[0306] An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of a hG-CSF with or withoutconjugation of the polypeptide to the polymer moiety. The rapid decreaseof hG-CSF serum concentrations has made it important to evaluatebiological responses to treatment with conjugated and non-conjugatedhG-CSF and variants thereof. Preferably, the conjugated andnon-conjugated hG-CSF and variants thereof of the present invention haveprolonged serum half-lives also after i.v. administration, making itpossible to measure by e.g. an ELISA method or by the primary screeningassay. Measurement of in vivo biological half-life was carried out asdescribed below.

[0307] Male Sprague Dawley rats (7 weeks old) were used. On the day ofadministration, the weights of the animals were measured (280-310 gramper animal). 100 μg per kg body weight of the non-conjugated andconjugated hG-CSF samples were each injected intravenously into the tailvein of three rats. At 1 minute, 30 minutes, 1, 2, 4, 6, and 24 hoursafter the injection, 500 μμl of blood was withdrawn from the eyes ofeach rat while under CO₂-anaesthesia. The blood samples were stored atroom temperature for 11 hours followed by isolation of serum bycentrifugation (4° C., 18000× g for 5 minutes). The serum samples werestored at −80° C. until the day of analysis. The amount of active G-CSFin the serum samples was quantified by the G-CSF in vitro activity assay(see primary assay 2) after thawing the samples on ice.

[0308] Another example of an assay for the measurement of in vivohalf-life of G-CSF or variants thereof is described in U.S. Pat. No.5,824,778, the content of which is hereby incorporated by reference.

[0309] Measurement of the in vivo Biological Activity in Healthy Rats ofConjugated and Non-Conjugated hG-CSF and Variants Thereof

[0310] Measurement of the in vivo biological effects of hG-CSF in SPFSprague Dawley rats (purchased from M & B A/S, Denmark) is used toevaluate the biological efficacy of conjugated and non-conjugated G-CSFand variants thereof.

[0311] On the day of arrival the rats are randomly allocated into groupsof 6. The animals are acclimatized for a period of 7 days whereinindividuals in poor condition or at extreme weights are rejected. Theweight range of the rats at the start of the acclimatization period is250-270 g.

[0312] On the day of administration the rats are fasted for 16 hoursfollowed by subcutaneous injection of 100 μg per kg body weight ofhG-CSF or a variant thereof. Each hG-CSF sample is injected into a groupof 6 randomized rats. Blood samples of 300 μl EDTA stabilized blood aredrawn from a tail vein of the rats prior to dosing and at 6, 12, 24, 36,48, 72, 96, 120 and 144 hours after dosing. The blood samples areanalyzed for the following haematological parameters: Haemoglobin, redblood cell count, haematocrit, mean cell volume, mean cell haemoglobinconcentration, mean cell haemoglobin, white blood cell count,differential leucocyte count (neutrophils, lymphocytes, eosinophils,basophils, monocytes). On the basis of these measurements the biologicalefficacy of conjugated and non-conjugated hG-CSF and variants thereof isevaluated. Further examples of assays for the measurement of in vivobiological activity of hG-CSF or variants thereof are described in U.S.Pat. Nos. 5,681,720, U.S. 5,795,968, U.S. 5,824,778, U.S. 5,985,265 andby Bowen et al., Experimental Hematology 27:425-432 (1999).

[0313] Measurement of the in vivo Biological Activity in Rats withChemotherapy-Induced Neutropenia of Conjugated and Non-Conjugated hG-CSFand Variants Thereof

[0314] SPF Sprague Dawley rats were purchased from M & B A/S, Denmark.On the day of arrival the rats are randomly allocated into groups of 6.The animals are acclimatized for a period of 7 days wherein individualsin poor condition or at extreme weights are rejected. The weight rangeof the rats at the start of the acclimatization period is 250-270 g. 24hours before administration of the hG-CSF samples the rats are injectedip. with 50 or 90 mg per kg body weight of cyclophosphamide (CPA). ThePEGylated hG-CSF variants are given as a single dose injected s.c. atday 0, while non-conjugated hG-CSF is injected s.c. either in a singledose at day 0 or on a daily basis. For hG-CSF or variants given in asingle dose at day 0, the dosage is 100 μg per kg body weight. Fornon-conjugated hG-CSF (Neupogen®) given on a daily basis, the dosagevaried and is given in the examples below. Each hG-CSF sample isinjected into a group of 6 randomized rats. Blood samples of 300 μl EDTAstabilized blood are drawn from a tail vein of the rats prior to dosingand at 6, 12, 24, 36, 48, 72, 96, 120, 144 and 168 hours after dosing.The blood samples are analyzed for the following haematologicalparameters: hemoglobin, red blood cell count, haematocrit, mean cellvolume, mean cell haemoglobin concentration, mean cell haemoglobin,white blood cell count, differential leucocyte count (neutrophils,lymphocytes, eosinophils, basophils, monocytes). On the basis of thesemeasurements the biological efficacy of conjugated and non-conjugatedhG-CSF and variants thereof is evaluated.

[0315] Determination of Polypeptide Receptor-Binding Affinity (On- andOff-Rate)

[0316] The strength of the binding between a receptor and ligand can bemeasured using an enzyme-linked immunoadsorption assay (ELISA), aradio-immunoassay (RIA), or other such immunodetection techniques wellknown in the art. The ligand-receptor binding interaction may also bemeasured with the Biacore® instrument, which exploits plasmon resonancedetection (Zbou et al., Biochemistry, 1993, 32, 8193-98; Faegerstram andO'Shannessy, 1993, In Handbook of Affinity Chromatography, 229-52,Marcel Dekker, Inc., NY).

[0317] The Biacore® technology allows one to bind receptor to a goldsurface and to flow ligand over it. Plasmon resonance detection givesdirect quantification of the amount of mass bound to the surface in realtime. This technique yields both on- and off-rate constants and thus aligand-receptor dissociation constant and an affinity constant can bedirectly determined.

[0318] In Vitro Immunogenicity Test of hG-CSF Conjugates

[0319] The reduced immunogenicity of a conjugate of the invention can bedetermined by use of an ELISA method measuring the immunoreactivity ofthe conjugate relative to a reference molecule or preparation. Thereference molecule or preparation is normally a recombinant human G-CSFpreparation such as Neupogen® or another recombinant human G-CSFpreparation, e.g. an N-terminally PEGylated rhG-CSF molecule asdescribed in U.S. Pat. No. 5,824,784. The ELISA method is based onantibodies from patients treated with one of these recombinant G-CSFpreparations. The immunogenicity is considered to be reduced when theconjugate of the invention has a statistically significant lowerresponse in the assay than the reference molecule or preparation.

[0320] Neutralisation of Activity in G-CSF Bioassay

[0321] The neutralisation of hG-CSF conjugates by anti-G-CSF sera isanalyzed using the G-CSF bioassay described above.

[0322] Sera from patients treated with the G-CSF reference molecule orfrom immunized animals are used. Sera are added either in a fixedconcentration (dilution 1:20-1:500 (pt sera) or 20-1000 ng/ml (animalsera)) or in five-fold serial dilutions of sera starting at 1:20 (ptsera) or 1000 ng/ml (animal sera). HG-CSF conjugate is added either inseven fold-dilutions starting at 10 nM or in a fixed concentration(1-100 pM) in a total volume of 80 μl DMEM medium +10% FCS. The sera areincubated for 1 hr. at 37° C. with hG-CSF conjugate.

[0323] The samples (0.01 ml) are then transferred to 96 well tissueculture plates containing NFS-60 cells in 0.1 ml DMEM media. Thecultures are incubated for 48 hours at 37° C. in a 5% CO₂ airatmosphere. 0.01 ml WST-1 (WST-1 cell proliferation agent, RocheDiagnostics GmbH, Mannheim, Germany) is added to the cultures andincubated for 150 min. at 37° C. in a 5% CO₂ air atmosphere. Thecleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenasesin viable cells results in the formation of formazan that is quantifiedby measuring the absorbance at 450 nm.

[0324] When hG-CSF conjugate samples are titrated in the presence of afixed amount of serum, the neutralising effect is defined as foldinhibition (FI) quantified as EC50(with serum)/EC50(without serum). Thereduction of antibody neutralisation of G-CSF variant proteins isdefined as$\left( {1 - \frac{\left( {{{FI}\quad {variant}} - 1} \right)}{\left( {{{FI}\quad {wt}} - 1} \right)}} \right) \times 100\%$

EXAMPLE 1

[0325] Construction and Cloning of Synthetic Genes Encoding hG-CSF

[0326] The following DNA fragments were synthesized following thegeneral procedure described by Stemmer et al. (1995), Gene 164, pp.49-53:

[0327] Fragment 1, consisting of a Bam HI digestion site, a sequenceencoding the YAP3 signal peptide (WO 98/32867), a sequence encoding theTA57 leader sequence (WO 98/32867), a sequence encoding a KEX2 proteaserecognition site (AAAAGA), a sequence encoding hG-CSF with its codonusage optimized for expression in E. coli, (SEQ ID NO:2) and a Xba Idigestion site.

[0328] Fragment 2, consisting of a Bam HI digestion site, a sequenceencoding the YAP3 signal peptide (WO 98/32867), a sequence encoding theTA57 leader sequence (WO 98/32867), a sequence encoding a histidine tag(SEQ ID NO:5), a sequence encoding a KEX2 protease recognition site(AAAAGA), a sequence encoding hG-CSF with its codon usage optimized forexpression in E. coli, (SEQ ID NO:2) and a Xba I digestion site.

[0329] Fragment 3, consisting of a Nde I digestion site, a sequenceencoding the OmpA signal peptide (SEQ ID NO:3), a sequence encodinghG-CSF with its codon usage optimized for expression in E. coli, (SEQ IDNO:2) and a Bam HI digestion site.

[0330] Fragment 4, consisting of a Bam HI digestion site, the Kozakconsensus sequence (Kozak, M. J Mol Biol 1987 Aug 20;196(4):947-50), asequence encoding the hG-CSF signal peptide (SEQ ID NO:7) and hG-CSFwith its codon usage optimized for expression in CHO cells (SEQ ID NO:8)and a Xba I digestion site.

[0331] DNA fragment 1 and 2 were inserted into the Bam HI and Xba Idigestion sites in plasmid pJSO37 (Okkels, Ann. New York Acad. Sci.782:202-207, 1996) using standard DNA techniques. This resulted inplasmids pG-CSFcerevisiae and pHISG-CSFcerevisiae.

[0332] DNA fragment 3 was inserted into the Nde I and Bam HI digestionsites in plasmid pET12a (Invitrogen) using standard DNA techniques. Thisresulted in plasmid pG-CSFcoli

[0333] DNA fragment 4 was inserted into the Bam HI and Xba I digestionsites in plasmid pcDNA3.1 (+) (Invitrogen) using standard DNAtechniques. This resulted in plasmid pG-CSFCHO.

EXAMPLE 2

[0334] Expression of hG-CSF in S. cerevisiae and E. coli

[0335] Transformation of Saccharomyces cerevisiae YNG318 (available fromthe American Type Culture Collection, VA, USA as ATCC 208973) witheither plasmid pG-CSFcerevisiae or pHISG-CSFcerevisiae, isolation oftransformants containing either of the two plasmids, and subsequentextracellular expression of hG-CSF without and with the HIS tag,respectively, was performed using standard techniques described in theliterature. Transformation of E. coli BL21 (DE3) (Novagen, Cat. No.69387-3) with pG-CSFcoli, isolation of transformants containing theplasmid and subsequent expression of hG-CSF in the supernatant and inthe periplasm of the cell was performed as described in the pET SystemManual (8^(th) edition) from Novagen.

[0336] Expression of hG-CSF by S. cerevisiae and E. coli was verified byWestern Blot analysis using the ImmunoPure Ultra-Sensitive ABC RabbitIgG Staining kit (Pierce) and a polyclonal antibody against hG-CSF(Pepro Tech EC Ltd.). It was observed that the protein had the correctsize.

[0337] The expression levels of hG-CSF with and without the N-terminalhistidine tag in S. cerevisiae and E. coli were quantified using acommercially available G-CSF specific ELISA kit (Quantikine Human G-CSFImmunoassay, R&D Systems Cat. No. DCS50). The measured values are listedbelow. Expression level Expression system (mg G-CSF per L) hG-CSF in S.cerevisiae 30 hG-CSF with histidine 25 tag in S. cerevisiae hG-CSF in E.coli 0.05

EXAMPLE 3

[0338] Generation of a Stable CHO-KI G-CSF Producer

[0339] The day before transfection the CHO K1 cell line (ATCC #CCl-61)is seeded in a T-25 flask in 5 ml DMEM/F-12 medium (Gibco # 31330-038)supplemented with 10% FBS and penicillin/streptomycin. The following day(at nearly 100% confluency) the transfection is prepared: 90 μl DMEMmedium without supplements is aliquoted into a 14 ml polypropylene tube(Coming). 10 μl Fugene 6 (Roche) is added directly into the medium andincubated for 5 min at room temperature. In the meantime 5 μg plasmidpG-CSFCHO is aliquoted into another 14 ml polypropylene tube. Afterincubation the Fugene 6 mix is added directly to the DNA solution andincubated for 15 min at room temperature. After incubation the wholevolume is added drop-wise to the cell medium.

[0340] The next day the medium is exchanged with fresh medium containing360 μg/ml hygromycin (Gibco). Every day hereafter the selection mediumis renewed until the primary transfection pool has reached 100%confluency. The primary transfection pool is sub-cloned by limiteddilution (300 cells seeded in five 96-well plates).

EXAMPLE 4

[0341] Purification of hG-CSF and variants thereof from S. cerevisiaeculture supernatants

[0342] Purification of hG-CSF was performed as follows:

[0343] Cells are removed by centrifugation. Cell depleted supernatant isthen filter sterilized through a 0.22 μm filter. Filter sterilizedsupernatant is diluted 5 fold in 10 mM sodium acetate pH 4.5. pH isadjusted by addition of 10 ml concentrated acetic acid per 5 liters ofdiluted supernatant. The ionic strength should be below 8 mS/cm beforeapplication to the cation exchange column.

[0344] Diluted supernatant is loaded at a linear flow rate of 90 cm/honto a SP-sepharose FF (Pharmacia) column equilibrated with 50 mM sodiumacetate, pH 4.5 until the effluent from the column reaches a stable UVand conductivity baseline. To remove any unbound material, the column iswashed using the equilibration buffer until the effluent from the columnreaches a stable level with respect to UV absorbance and conductivity.The bound G-CSF protein is eluted from the column using a lineargradient; 30 column volumes; 0-80% buffer B (50 mM NaAc, pH 4.5, 750 mMNaCl) at a flow rate of 45 cm/h. Based on SDS-polyacryl amide gelelectrophoresis, fractions containing G-CSF are pooled. Sodium chlorideis added until the ionic strength of the solution is more than 80 mS/cm.

[0345] The protein solution is applied onto a Phenyl Toyo Pearl 650Scolumn equilibrated with 50 mM NaAc, pH 4.5, 750 mM NaCl. Any unboundmaterial is washed off the column using the equilibration buffer.Elution of G-CSF is performed by applying a step gradient of MilliQwater. Fractions containing G-CSF are pooled. By using this 2-step downstream processing strategy, more than 90% pure G-CSF can be obtained.The purified protein is then quantified using spectrophotometricmeasurements at 280 nm and/or by amino acid analysis.

[0346] Fractions containing G-CSF are pooled. Buffer exchange andconcentration is performed using VivaSpin concentrators (mwco: 5 kDa).

EXAMPLE 5

[0347] Identification and Quantification of Non-Conjugated andConjugated hG-CSF and Variants Thereof

[0348] SDS-Polyacryl Amide Gel Electrophoresis

[0349] The purified, concentrated G-CSF was analyzed by SDS-PAGE. Asingle band having an apparent molecular weight of approx. 17 kDa wasdominant.

[0350] Absorbance

[0351] An estimate of the G-CSF concentration is obtained byspectrophotometric methods. By measuring the absorbance at 280 nm andusing a theoretically extinction coefficient of 0.83, the proteinconcentration can be calculated.

[0352] Amino Acid Analysis

[0353] A more accurate protein determination can be obtained by aminoacid analysis. Amino acid analysis performed on a purified G-CSFrevealed that the experimentally determined amino acid composition is inagreement with the expected amino acid composition based on the DNAsequence.

EXAMPLE 6

[0354] MALDI-TOF Mass Spectrometry of PEGylated wt G-CSF and G-CSFVariants

[0355] MALDI-TOF mass spectrometry was used to evaluate the number ofPEG-groups attached to PEGylated wt G-CSF and to selected PEGylatedG-CSF variants.

[0356] Wt G-CSF contains 5 primary amines that are the expectedattachment sites for SPA-PEG (the N-terminal amino-group and theε-amino-group on K16, K23, K34 and K40). Following PEGylation of wtG-CSF with SPA-PEG 5000, MALDI-TOF mass spectrometry showed the presenceof species of wt G-CSF with mainly 4, 5 and 6 PEG-groups attached. Inaddition, wt G-CSF with 7 PEG-groups attached was clearly seen althoughin minor amounts.

[0357] The G-CSF variant having the substitutions K16R, K34R, K40R,Q70K, Q90K, and Q120K also contains 5 primary amines (the N-terminalamino-group and the ε-amino-group on K23, K70, K90 and K120). FollowingPEGylation of this G-CSF variant with SPA-PEG5000, MALDI-TOF massspectrometry showed the presence of species of the G-CSF variant withmainly 4, 5 and 6 PEG-groups attached. In addition, the G-CSF variantwith 7 PEG-groups attached was clearly seen although in minor amounts.

[0358] The G-CSF variant having the substitutions K16R, K34R, and K40Rcontains 2 primary amines (the N-terminal amino-group and theε-amino-group on K23). Following PEGylation of this G-CSF variant withSPA-PEG 12000, MALDI-TOF mass spectrometry showed the presence ofspecies of the G-CSF variant with mainly 2 and 3 PEG-groups attached. Inaddition, the G-CSF variant with 4 PEG-groups attached was clearly seenalthough in minor amounts.

[0359] These observations clearly show that in addition to amino acidresidues containing amine groups, other amino acid residues aresometimes PEGylated under the PEGylation conditions used. It also showsthat it is of some importance for the PEGylation where amine groups areintroduced. This has also been observed using SDS-PAGE analysis of wtG-CSF and G-CSF variants.

[0360] As described in Example 12, it has been shown that histidine 170is fully PEGylated when the SPA-PEG chemistry is used. Furthermore, K23and S159 are partly PEGylated. This explains the presence of 1-2 extraPEGylation sites besides the primary amines in hG-CSF and the variantsthat have been made.

EXAMPLE 7

[0361] Peptide Mapping of PEGylated and Non-PEGylated G-CSF Variants

[0362] In order to map the additional attachment sites for SPA-PEG onG-CSF and G-CSF variants the following strategy was used.

[0363] A G-CSF variant with a low number of amine groups was chosen inorder to reduce the number of expected PEGylation sites to a minimum.The G-CSF variant chosen has the substitutions K16R, K34R, K40R andH170Q. Apart from the 1-amino-group on K23 that previous data had shownnot to be PEGylated to any large extent, this variant only contains oneprimary amine at the N-terminal. Thus, the background PEGylation onamine groups is significantly reduced in this G-CSF variant. The G-CSFvariant was PEGylated using SPA-PEG 5000. Following PEGylation, theG-CSF variant was denatured, the disulphide bonds reduced, the resultingthiol groups alkylated, and the alkylated and PEGylated protein degradedwith a glutamic acid-specific protease. Finally, the resulting peptideswere separated by reversed phase HPLC.

[0364] Parallel with this, the non-PEGylated version of the G-CSFvariant with the substitutions K16R, K34R, and K40R was treatedidentically in order to create a reference HPLC chromatogram.

[0365] Comparison of the HPLC chromatograms of the degradation of thePEGylated G-CSF variant and the non-PEGylated G-CSF variant should thenreveal which peptides disappear upon PEGylation. Identification of thesepeptides by N-terminal amino acid sequencing of the peptide from thenon-PEGylated G-CSF variant then indirectly points to the positions thatare PEGylated.

[0366] In principle, it would have been preferable to use thenon-PEGylated version of the G-CSF variant having all the substitutionsK16R, K34R, K40R and H170Q, but for all practical purposes this does notmatter.

[0367] More specifically, approximately 1 mg of the PEGylated G-CSFvariant having the substitutions K16R, K34R, K40R and H170Q andapproximately 500 μg of the non-PEGylated G-CSF variant having thesubstitutions K16R, K34R, and K40R were dried in a SpeedVacconcentrator. The two samples were each dissolved in 400 μl 6 Mguanidinium, 0.3 M Tris-HCl, pH 8.3 and denatured overnight at 37° C.Following denaturation, the disulfide bonds in the proteins were reducedby addition of 50 μl 0.1 M DTT in 6 M guanidinium, 0.3 M Tris-HCl, pH8.3. After 2 h of incubation at ambient temperature the thiol groupspresent were alkylated by addition of 50 μl 0.6 M iodoacetamid in 6 Mguanidinium, 0.3 M Tris-HCl, pH 8.3. Alkylation took place for 30 min atambient temperature before the reduced and alkylated proteins werebuffer changed into 50 mM NH₄HCO₃ using NAP5 columns. The volumes of thesamples were reduced to approximately 200 μl in a SpeedVac concentratorbefore addition of 20 μg and 10 μg glutamic acid-specific protease,respectively. The degradations with glutamic acid-specific protease werecarried out for 16 h at 37° C. The resulting peptides were separated byreversed phase HPLC employing a Phenomenex Jupiter C₁₈ column (0.2 * 5cm) eluted with a linear gradient of acetonitrile in 0.1% aqueous TFA.The collected fractions were analyzed by MALDI-TOF mass spectrometry andsubsequently selected peptides were subjected to N-terminal amino acidsequence analysis.

[0368] Comparison of the HPLC chromatograms of the degradations of thePEGylated G-CSF variant and the non-PEGylated G-CSF variant revealedthat only two fractions disappear upon PEGylation. N-terminal amino acidsequence analysis of the two fractions from the non-PEGylated G-CSFvariant showed that the peptides both were derived from the N-terminalof G-CSF. One peptide consisted of amino acid residues 1-11 generated byan unexpected cleavage following Gln11. The other peptide consisted ofamino acid residues 1-19 generated by an expected cleavage followingGlu19.

[0369] It was expected that the N-terminal peptide of G-CSF would beidentified using this approach, as the N-terminal amino group is easilyPEGylated. However, none of the additional attachment sites for SPA-PEG5000 were identified using this approach.

[0370] An alternative to the indirect identification of PEG 5000attachment sites is direct identification of the attachment sites inPEGylated peptides. However, the fractions containing the PEGylatedpeptides in the HPLC separation of the degraded PEGylated G-CSF variantare poorly separated from each other and from several fractionscontaining non-PEGylated peptides. Thus, N-terminal amino acid sequenceanalysis of these fractions did not result in any useful data except foran indication that K23 could be partially PEGylated.

[0371] To overcome these problems, two pools of PEGylated peptides weremade from the fractions from the first HPLC separation. These two poolswere dried in a SpeedVac concentrator, dissolved in 200 μl freshlyprepared 50 mM NH₄HCO₃ and further degraded with 1 μg of chymotrypsin.The resulting peptides were separated by reversed phase HPLC employing aPhenomenex Jupiter C,8 column (0.2*5 cm) eluted with a linear gradientof acetonitrile in 0.1% aqueous TFA. The collected fractions wereanalyzed by MALDI-TOF mass spectrometry and subsequently selectedpeptides were subjected to N-terminal amino acid sequence analysis.

[0372] From the N-terminal amino acid sequence determinations it couldbe determined that K23 as well as S159 are partially PEGylated. It wasnot possible to determine the exact degree of PEGylation at these twopositions, but the PEGylation is only partial as peptides where K23 andS1159 are unmodified were identified and sequenced from the initial HPLCseparation.

EXAMPLE 8

[0373] Glycosylation of wt G-CSF and G-CSF Variants

[0374] A consistent observation when analyzing purified wt G-CSF andG-CSF variants by MALDI-TOF mass spectrometry is the presence of anadditional component with a mass approximately 324 Da larger than themass of the G-CSF molecule analyzed. As the component with the lowestmass invariantly has the mass of the G-CSF molecule and because theG-CSF molecules have the correct N-terminal amino acid sequence, it wasconcluded that the additional component is a modified G-CSF moleculecarrying two hexose residues. In many cases the unmodified G-CSFmolecule gives rise to the most intense signal but in some cases theintensity of the signal for the modified G-CSF molecule is the mostintense.

[0375] During the analysis of the peptides generated with the aim ofidentifying the additional PEGylation sites, two peptides of interestfor identifying the site of glycosylation were identified in each of thedegradations.

[0376] In both HPLC separations, the two peptides elute next to eachother and MALDI-TOF mass spectrometry shows a mass difference betweenthe two peptides of approximately 324 Da. The mass spectrometry dataindicates that the peptide covers amino acid residues 124-162.N-terminal amino acid sequence analysis of all four peptides showed thatthis assignment is correct and that Thr133 is the only site ofmodification. In the peptides with the mass of the unmodified peptide,Thr133 is clearly seen in the sequence, while no amino acid residue canbe assigned at position 133 in the peptides with an additional mass of324 Da. As all other amino acid residues could be assigned in thesequence, it was concluded that Thr133 is the only site of modification.This glycosylation site was previously reported to be used inrecombinant G-CSF expressed in CHO cells, although the glycan isdifferent from the one attached by yeast.

[0377] The non-glycosylated wt G-CSF has been separated from theglycosylated wt G-CSF, employing reversed phase HPLC using a Vydac C₁₈column (0.21*5 cm) isocratically eluted with 51% acetonitrile in 0.1%TFA, as a fraction shown by MALDI-TOF mass spectrometry only to containthe non-glycosylated form of wt G-CSF.

EXAMPLE 9

[0378] Separation of G-CSF Molecules with Different Numbers of PEGMolecules Covalently Attached

[0379] Separation of G-CSF molecules covalently attached to 4, 5 or 6PEG-groups was obtained as follows. PEGylated protein in 20 mM sodiumcitrate, pH 2.5 was applied to an SP-sepharose FF column equilibratedwith 20 mM sodium citrate pH 2.5. Any unbound material was washed offthe column. Elution was performed using a pH gradient. PEGylated G-CSFbegan to elute from the column at approx. pH 3.8 and continued to elutein fractions covering a pH span from 3.8 to 4.5.

[0380] The fractions were subjected to SDS-PAGE and mass spectrometricanalysis. These analyses indicate that G-CSF having the highest degreeof PEGylation is located in the “low pH fractions”. PEGylated G-CSFhaving a lower degree of PEGylation is eluted in the “high pHfractions”.

[0381] Amino acid analysis performed on PEGylated G-CSF showed goodconsistency between the theoretically and the experimentally determinedextinction coefficient.

EXAMPLE 10

[0382] Construction of hG-CSF Variants

[0383] Specific substitutions of existing amino acids in hG-CSF to otheramino acid residues, e.g. the specific substitutions discussed above inthe general description, were introduced using standard DNA techniquesknown in the art. The new G-CSF variants were made using plasmidpG-CSFcerevisiae containing the gene, encoding hG-CSF without the HIStag, as DNA template in the PCR reactions. The variants were expressedin S. cerevisiae and purified as described in Example 4. Some of theconstructed G-CSF variants are listed below (see Examples 12 and 13).

EXAMPLE 11

[0384] Covalent Attachment of SPA-PEG to hG-CSF or Variants Thereof

[0385] Human G-CSF and variants thereof were covalently linked toSPA-PEG 5000, SPA-PEG 12000 and SPA-PEG 20000 (Shearwater) as describedabove (“PEGylation of hG-CSF and variants thereof in solution”). The invitro activities of the conjugates are listed in Example 13.

EXAMPLE 12

[0386] Identification of SPA-PEG Attachment Sites in G-CSF bySite-Directed Mutagenesis Followed by PEGylation of the PurifiedVariants

[0387] SPA-PEG may be attached to other amino acid residues than lysinein G-CSF. In order to determine whether SPA-PEG was attached tohistidines, serines, threonines and arginines, variants were made inwhich these amino acids were substituted to lysine, alanine orglutamine. The variants were expressed in S. cerevisiae, purified andPEGylated followed by analysis of the number of attached SPA-PEGmolecules on SDS-PAGE. This analysis was performed by visual inspectionof the SDS-PAGE gels, all of which contained three major bands. Thedegree of PEGylation was estimated to the nearest 5% for each band basedon the relative size of the bands. A reduction in the number of attachedSPA-PEG molecules after substitution of a given amino acid withglutamine or alanine strongly indicates that this amino acid isPEGylated by SPA-PEG, and this observation is further supported by anunchanged degree of PEGylation after substitution of the amino acid tolysine. The analyzed variants are listed below. G-CSF variant No. ofattached PEG groups hG-CSF 10% 4 PEG, 75% 5 PEG, 15% 6 PEG K23R 10% 4PEG, 85% 5 PEG, 5% 6 PEG H43Q 10% 4 PEG, 75% 5 PEG, 15% 6 PEG H43K 10% 5PEG, 75% 6 PEG, 15% 7 PEG H52Q 10% 4 PEG, 75% 5 PEG, 15% 6 PEG H52K 10%5 PEG, 75% 6 PEG, 15% 7 PEG H156Q 10% 4 PEG, 75% 5 PEG, 15% 6 PEG H156K10% 5 PEG, 75% 6 PEG, 15% 7 PEG H170Q 10% 3 PEG, 75% 4 PEG, 15% 5 PEGH170K 10% 4 PEG, 75% 5 PEG, 15% 6 PEG K16/34R 10% 2 PEG, 75% 3 PEG, 15%4 PEG K16/34R R22K 10% 3 PEG, 75% 4 PEG, 15% 5 PEG K16/34R R22Q 10% 2PEG, 75% 3 PEG, 15% 4 PEG K16/34R S142A 10% 2 PEG, 75% 3 PEG, 15% 4 PEGK16/34/40R 10% 1 PEG, 75% 2 PEG, 15% 3 PEG K16/34/40R S53K 10% 2 PEG,75% 3 PEG, 15% 4 PEG K16/34/40R S53A 10% 2 PEG, 75% 3 PEG, 15% 4 PEGK16/34/40R S62K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S66K 10% 2PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S80K 10% 2 PEG, 75% 3 PEG, 15% 4PEG K16/34/40R T105K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R T133K10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S142K 10% 2 PEG, 75% 3 PEG,15% 4 PEG K16/34/40R R147K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40RS155K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S159K 10% 2 PEG, 85% 3PEG, 5% 4 PEG K16/34/40R S170K 10% 1 PEG, 75% 2 PEG, 15% 3 PEG

[0388] The data show that besides the N-terminus, K16, K34 and K40,SPA-PEG also is covalently bound to H170. Furthermore, the data showthat only 10% of the available K23 amino acid residues are PEGylated,and that approximately 10% of S159 is PEGylated.

EXAMPLE 13

[0389] In vitro Biological Activity of Non-Conjugated and ConjugatedhG-CSF and Variants Thereof

[0390] The in vitro biological activities of conjugated andnon-conjugated hG-CSF and variants thereof were measured as describedabove in “Primary assay 2—in vitro hG-CSF activity assay”. The in vitrobioactivities, represented by the measured EC50 values for each variantwith and without conjugation of SPA-PEG 5000 to the available PEGylationsites, are listed below. The values have been normalized with respect tothe EC50 value of non-conjugated hG-CSF (Neupogen®), i.e. the values inthe table indicate % activity relative to the activity of non-conjugatedhG-CSF. This value was measured simultaneously with the variants eachtime under identical assay conditions. The EC50 value of hG-CSF in thedescribed assay is 30 pM. EC50 (% of hG-CSF) EC50 (% of hG-CSF)conjugated to G-CSF variant non-conjugated SPA-PEG 5000 G-CSF withN-terminal Histidine tag 10 Not determined G-CSF without N-terminalHistidine tag 100 0.1 16R 100 1 16Q 80 1 23Q 80 0.1 23R 100 0.1 34R 1001 34A 80 1 34Q 70 1 40R 50 1 K16/23R 100 1 K16/23Q 80 1 K34/40R 50 5K16/34R 100 10 K16/40R 50 5 K16/23/34R 50 10 K16/23/40R 50 5 K16/34/40R35 30 K16/23/34/40R 20 15 K16/34/40R L3K 50 25 K16/34/40R E45K Expressedat low levels Not determined K16/34/40R E46K 10 1 K16/34/40R S53K 5 0.5K16/34/40R S62K 10 0.5 K16/34/40R S66K 20 2 K16/34/40R Q67K 10 0.2K16/34/40R Q70K 30 20 K16/34/40R S76 50 20 K16/34/40R Q77 1 0 K16/34/40RS80K 10 0.2 K16/34/40R Q90K 30 20 K16/34/40R E98K Expressed at lowlevels Not determined K16/34/40R D104K 10 0.9 K16/34/40R T105K 30 10K16/34/40R Q120K 30 20 K16/34/40R Q131K Expressed at low levels Notdetermined K16/34/40R T133K 30 10 K16/34/40R Q134K 10 0.2 K16/34/40RS142K 20 7 K16/34/40R R147K 20 1 K16/34/40R S155K 20 1 K16/34/40R Q15820 5 K16/34/40R S159K 20 3 K16/34/40R Q70K Q90K Not determined 20K16/34/40R Q70K Q120K 25 25 K16/34/40R Q90K T105K 40 10 K16/34/40R Q90KQ120K 25 15 K16/34/40R Q90K S159K 45 Not determined K16/34/40R T105KQ120K 20 8 K16/34/40R T105K S159K 40 20 K16/34/40R Q120K T133K 20 8K16/34/40R Q120K S142K 10 2 K16/34/40R Q70K Q90K T105K 10 4 K16/34/40RQ70K Q90K Q120K 20 12 K16/34/40R Q70K Q90K T133K 15 5 K16/34/40R Q70KT105K Q120K 10 2 K16/34/40R Q70K Q120K T133K 15 2 K16/34/40R Q70K Q120KS142K 10 1 K16/34/40R Q90K T105K Q120K 10 2 K16/34/40R Q90K T105K T133K10 2 K16/34/40R Q90K T105K S159K 55 5 K16/34/40R Q90K Q120K T133K 15 2K16/34/40R Q90K Q120K S142K 10 1 K16/34/40R T105K Q120K T133K 10 1K16/34/40R Q120K T133K S142K 10 1

[0391] The data show that substitution of K23 to arginine does notincrease the activity of the conjugated protein. This is due to the factthat only 10% of K23 is PEGylated, whereby the conjugated K23R varianthas essentially the same number of PEG groups attached to it and has sthe same location of the PEGylation sites as hG-CSF. Removal of theremaining lysines at position K16, K34 and K40 resulted in a G-CSFvariant with significant activity after PEGylation. Conjugation ofSPA-PEG 5000 to this variant does not decrease the activitysignificantly as compared to the non-conjugated variant. Thus,PEGylation of the N-terminus and H170 with SPA-PEG 5000 (see Example 12)does not decrease the activity of hG-CSF. It was decided to use hG-CSFK16R K34R K40R as the backbone for insertion of new PEGylation sites. 24new PEGylation sites between residues L3 and H159 were introduced inthis backbone. These residues are distributed over the parts of hG-CSFthat do not interact with the G-CSF receptor. Introduction of newPEGylation sites at positions L3, Q70, S76, Q90, T105, Q120, T133 andS142 resulted in hG-CSF variants that retained a significant amount ofactivity after PEGylation by SPA-PEG 5000. Thus, some of these newPEGylation sites were combined in hG-CSF variants that had 2 or 3 newPEGylation sites.

[0392] Furthermore, SPA-PEG 12000 and SPA-PEG 20000 were attached to agroup a selected hG-CSF variants. The in vitro activities are listedbelow (% of Neupogen®).

[0393] EC50 (% of hG-CSF) EC50 (% of hG-CSF) EC50 (% of hG-CSF) EC50(%of hG-CSF) conjugated to conjugated to G-CSF variant SPA-PEG 12000SPA-PEG 20000 K16/34/40R 10 1 K16/34/40R Not determined 7 Q90KK16/34/40R 8 Not determined Q70K Q90K K16/34/40R 1 <1 Q90K T105KK16/34/40R 6 5 T105K S159K K16/34/40R 1 <1 Q90K T105K S159K

EXAMPLE 14

[0394] In vivo Half-Life of Non-Conjugated and Conjugated hG-CSF andVariants Thereof

[0395] The in vivo half-lives of non-conjugated hG-CSF (Neupogen®),SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K andSPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90K T105K S159K weremeasured as described above (“Measurement of the in vivo half-life ofconjugated and non-conjugated rhG-CSF and variants thereof”). Theresults are shown in FIGS. 1 and 2. The in vivo half-life of Neupogen®was determined to be 2.01 hours and 1.40 hours, respectively. In anearlier, similar experiment (U.S. Pat. No. 5,824,778), the in vivohalf-life of hG-CSF was determined to be 1.79 hours. The results of theexperiments described herein can therefore be directly compared to thatexperiment. The in vivo half-lives of SPA-PEG 5000 conjugated hG-CSFK16R K34R K40R Q70K Q90K Q120K and SPA-PEG 5000 conjugated hG-CSF K16RK34R K40R Q90K T105K S159K were determined to be 12.15 hours and 16.10hours, respectively. Thus, introducing new PEGylation sites in hG-CSFand conjugating SPA-PEG 5000 to them has resulted in a significantincrease in the in vivo half-life.

[0396] In the earlier experiment described above (U.S. Pat. No.5,824,778), the in vivo half-life of hG-CSF conjugated to a largerN-terminally attached PEG molecule (10 kDa) was determined to be 7.05hours. Thus, SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90KQ120K and SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90K T105K S159Khave significantly longer half-lives than both Neupogen® and hG-CSF witha 10 kDa N-terminally conjugated PEG molecule. SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q90K Q120K and SPA-PEG 5000 conjugated hG-CSFK16R K34R K40R Q90K T105K S159K both have three removed endogenousPEGylation sites and three new introduced PEGylation sites and thus areidentical in size. The only difference between the two compounds is thein vitro activity, which is 12% and 5%, respectively, of that ofNeupogen®D. This difference results in a longer in vivo half-life ofSPA-PEG 5000 conjugated K16R K34R K40R Q90K T105K S 159K compared toSPA-PEG 5000 conjugated K16R K34R K40R Q70K Q90K Q120K. Since the invitro activities correlate with the receptor binding affinities of thecompounds, it can be concluded that the receptor-mediated clearance ofSPA-PEG 5000 conjugated K16R K34R K40R Q90K T105K S159K is slower thanthat of SPA-PEG 5000 conjugated K16R K34R K40R Q70K Q90K Q120K. Thus, acombination of increasing the size and reducing the in vitro activity ofG-CSF results in G-CSF compounds with significantly longer in vivohalf-lives than previously described compounds.

EXAMPLE 15

[0397] In vivo Biological Activity in Healthy Rats of Non-Conjugated andConjugated hG-CSF and Variants Thereof

[0398] The in vivo biological activities of non-conjugated hG-CSF(Neupogen®), SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K,SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K T133K and SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q90K Q120K T133K were measured asdescribed above (“Measurement of the in vivo biological activity inhealthy rats of conjugated and non-conjugated hG-CSF and variantsthereof”). The results are shown in FIGS. 3 and 4.

[0399] No activity of Neupogen® could be detected at 48 hours afterinjection of 100 μg per kg body weight at t=0 hours. Activity of SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K, SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q120K T133K and SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q90K Q120K T133K could be detecteduntil 72 hours after the initial injection, while SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K remained active in vivountil 96 hours after the initial injection. Thus, it was shown that allof these conjugated variants had a longer in vivo biological activitythan Neupogen® and that SPA-PEG 5000 conjugated hG-CSF K16R K34R K40RQ70K Q90K Q120K remained active twice as long in vivo as Neupogen®.SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q120KT133K andSPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90K Q120K T133K, bothwith an in vitro activity of 2% of Neupogen® (Example 13), did notinduce the same level of white blood cell formation during the initial12 hours after administration as observed after administration ofNeupogen®, SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K andSPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K. Thus, thecompounds with an in vitro activity of 2% or less compared to that ofNeupogen® were unable to stimulate full formation of white blood cellsimmediately after administration.

[0400] Furthermore, the in vivo biological activities of Neupogen®,SPA-PEG 12000 conjugated hG-CSF K16R K34R K40R and different doses ofSPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K weremeasured as described above (“Measurement of the in vivo biologicalactivity in healthy rats of conjugated and non-conjugated hG-CSF andvariants thereof”). The results are shown in FIG. 5. As observedearlier, no activity of Neupogen® could be detected 48 hours after theinitial injection of 100 μg per kg body weight. Administration of 5 μgper kg body weight of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70KQ90K Q120K resulted in a slightly longer in vivo biological activitythan Neupogen®, while administration of 25 μg per kg body weight and 100μg per kg body weight of this compound resulted in hG-CSF activity until72 and 96 hours, respectively, after the initial injection. Thus, theduration of action of the SPA-PEG conjugated hG-CSF compounds can becontrolled by increasing or decreasing the standard dosing regimen.SPA-PEG 12000 conjugated hG-CSF K16R K34R K40R remained active in vivountil 72 hours after administration of 100 μg per kg body weight. Asdescribed in Example 6, SPA-PEG 12000 conjugated hG-CSF K16R K34R K40Rhas 2 or 3 SPA-PEG 12000 groups attached while SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q90K Q120K has 5 or 6 SPA-PEG 5000 groupsattached. Thus, the molecular weights of the two compounds are 42-54 kDaand 43-48 kDa, respectively. The in vitro activities of the twocompounds are 30% and 12%, respectively, of that of Neupogen®. Thelonger in vivo biological activity of SPA-PEG 5000 conjugated hG-CSFK16R K34R K40R Q70K Q90K Q120K as compared to SPA-PEG 12000 conjugatedhG-CSF K16R K34R K40R with essentially the same molecular weightsuggests that when the size of the G-CSF compounds is increased above acertain molecular weight through PEGylation, the duration of action canonly be increased further by reducing the specific activity of the G-CSFcompounds and thus, the receptor-mediated clearance (see Example 14).

[0401] Furthermore, the in vivo biological activities of Neupogen®,SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG5000 conjugated hG-CSF K16R 34R 40R Q90K T105K S159K and SPA-PEG 20000conjugated hG-CSF K16R 34R 40R T105K S159K were measured as describedabove (“Measurement of the in vivo biological activity in healthy ratsof conjugated and non-conjugated hG-CSF and variants thereof”). Theresults are shown in FIG. 6.

[0402] As observed earlier, the conjugated hG-CSF variants had asignificant longer duration of action than Neupogen®. Administration ofeach of these three conjugated hG-CSF variants resulted in formation ofwhite blood cells at the same rate and to the same level as observedafter administration of Neupogen® during the initial 12 hours afteradministration. The in vitro activities of SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG 5000 conjugated hG-CSFK16R 34R 40R Q90K T105K S159K and SPA-PEG 20000 s1 conjugated hG-CSFK16R 34R 40R T105K S159K are 12%, 5% and 5%, respectively, of that ofNeupogen®, and thus, a hG-CSF compound with 5% of Neupogen® activity invitro is able to induce full white blood cell formation afteradministration.

[0403] The apparent size on SDS-PAGE of Neupogen®, SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG 5000conjugated hG-CSF K16R 34R 40R Q90K T105K S159K and SPA-PEG 20000conjugated hG-CSF K16R 34R 40R T105K S159K is 18 kDa, 60 kDa, 60 kDaand >100 kDa, respectively. SPA-PEG 5000 conjugated hG-CSF K16R 34R 40RQ90K T105K S159K and SPA-PEG 20000 conjugated hG-CSF K16R 34R 40R T105KS159K have almost identical durations of action in vivo, showing thatthe duration of action is not increased by increasing the molecular sizeof the conjugated hG-CSF compounds above an apparent size of about 60kDa. Instead, when the apparent size of the conjugated hG-CSF compoundsis above about 60 kDa, the duration of action may be increased bereducing the in vitro activity and hence, the receptor binding affinityof the compound. An additional example of this (see above) can beobserved by comparing the in vivo duration of action of SPA-PEG 5000conjugated hG-CSF K16R 34R 40R Q70K Q90K Q120K and SPA-PEG 5000conjugated hG-CSF K16R34R4OR Q90K T105KS159K. The two compounds bothhave an apparent size of 60 kDa, while the in vitro activities are 12%and 5%, respectively. This difference is reflected directly in the invivo duration of action of the two compounds, which is 96 hours and 144hours, respectively.

EXAMPLE 16

[0404] In vivo Biological Activity in Rats with Chemotherapy-InducedNeutropenia of Non-Conjugated and Conjugated hG-CSF and Variants Thereof

[0405] The in vivo biological activities in rats withchemotherapy-induced neutropenia of non-conjugated hG-CSF (Neupogen®),SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K andSPA-PEG 20000 conjugated hG-CSF K16R K34R K40R Q90K were measured asdescribed above (“Measurement of the in vivo biological activity in ratswith chemotherapy-induced neutropenia of conjugated and non-conjugatedhG-CSF and variants thereof”) using 50 mg per kg body weight of CPA anda single dose (100 μg per kg body weight) of G-CSF. The results areshown in FIG. 7. The three compounds induced an initial formation ofwhite blood cells with identical rates. Thus, an in vitro activity of 4%of that of Neupogen® is sufficient for a conjugated hG-CSF compound togive full stimulation of white blood cell formation in vivo immediatelyafter administration. After 36 hours the number of white blood cells(WBC) in the Neupogen®-treated rats dropped to the level that wasobserved in the untreated group (<3×10⁹ cells per liter). At this pointthe rats were neutropenic. The level of WBC in both groups reachednormal levels (9×10⁹ cells per liter) after 144 hours.

[0406] The level of WBC in the two groups treated with SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K and SPA-PEG 20000conjugated hG-CSF K16R K34R K40R Q90K dropped to a minimum of 4×10⁹cells per liter after 48 hours and then immediately started to increase.The WBC levels in both groups were back to normal after 96 hours. Thus,the two conjugated hG-CSF compounds were able to both relieve the degreeof neutropenia and to reduce the time until the WBC levels were back tonormal (the duration of neutropenia) significantly from 112 hours in theNeupogen®-treated group to 48 hours in the groups treated with eitherSPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K andSPA-PEG 20000 conjugated hG-CSF K16R K34R K40R Q90K. SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K more efficientlyshortened the duration of neutropenia as compared to SPA-PEG 20000conjugated hG-CSF K16R K34R K40R Q90K. Since the apparent size of bothmolecules is above 60 kDa (60 kDa and 80 kDa, respectively) this cannotbe explained by a lower renal clearance of SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q90K T105K than SPA-PEG 20000 conjugatedhG-CSF K16R K34R K40R Q90K. The in vitro activity of SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K and SPA-PEG 20000conjugated hG-CSF K16R K34R K40R Q90K are 4% and 7% of Neupogen®,respectively. This means that the receptor binding affinity and thus,the receptor-mediated clearance, of SPA-PEG 5000 conjugated hG-CSF K16RK34R K40R Q70K Q90K T105K is lower than for SPA-PEG 20000 conjugatedhG-CSF K16R K34R K40R Q90K in the initial 48 hours after administrationwhere the white blood cell levels are increased. Hence, when the ratsbecome neutropenic after 48 hours, the in vivo concentration of SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K is higher thanSPA-PEG 20000 conjugated hG-CSF K16R K34R K40R Q90K. Since a relativelylow in vitro G-CSF activity of 4-5% of that of Neupogen® is sufficientto obtain full activation of the G-CSF receptors on the neutrophilprogenitor cells (see above), this higher G-CSF concentration after 48hours explains the faster increase in WBC levels in the SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K-treated group. Thus, inrats with chemotherapy-induced neutropenia, a conjugated G-CSF compoundof the invention with an apparent size of at least about 60 kDa and anin vitro activity of 4% of that of Neupogen® is superior to similar sizecompounds with a higher in vitro activity.

EXAMPLE 17

[0407] Purification of G-CSF from S. cerevisiae Culture Supernatants

[0408] This example provides an alternative purification procedure tothat of Example 4 for purification of hG-CSF and G-CSF variants.

[0409] Cells are removed by centrifugation, 5000 rpm, 10 min, 4° C., andthe clarified supernatant is filtered through a 0.22 μm filter. Theclarified and filtered supernatant is concentrated and diafiltered into50 mM sodium acetate, pH 4.5, by Tangential Flow Filtration using 10 kDamembranes.

[0410] The resulting ultra filtrate is applied onto an SP-sepharosecolumn (200 ml packed bed) equilibrated with at least 5 column volumesof 50 mM sodium acetate. Samples are loaded at a flow rate of approx. 20ml/min. The column is washed using the equilibration buffer until astable effluent is obtained as determined by absorbance at 280 nm. Usinga stepwise buffer gradient (e.g. 10%, 20%, 30% and 35% buffer), G-CSF iseluted at 35% buffer at ambient flow rate, where the buffer is 750 mMNaCl in 50 mM sodium acetate.

[0411] This one-step method yields >95% pure G-CSF (as determined bySDS-PAGE).

EXAMPLE 18

[0412] Separation of Multi-PEGylated Species of G-CSF

[0413] Example 9 above describes a method for separation of G-CSFmolecules with different numbers of PEG groups attached. This exampleprovides an alternative procedure for separation of such multi-PEGylatedG-CSF species in order to obtain a G-CSF product with a desired degreeof uniformity in terms of the number of attached PEG groups.

[0414] A mixture of PEGylated G-CSF, covalently linked to e.g. SPA-PEG5000 (Shearwater) as described above (“PEGylation of hG-CSF and variantsthereof in solution”), is diluted with 20 mM citrate buffer, pH 2.5. Theconductivity should be <3.5 mS/cm. The pH is adjusted to 2.5 asnecessary using dilute HCl. The following buffers are used for theseparation:

[0415] Buffer A: 20 mM sodium citrate, pH 2.5 (equilibration and washingbuffer).

[0416] Buffer B: 20 mM sodium citrate, pH 2.5; 750 mM sodium chloride(elution buffer)

[0417] The sample to be separated is loaded onto an equilibratedSP-sepharose HP column (7 ml) at a flow rate of 2 ml/min. The column iswashed with Buffer A until a stable baseline is obtained as monitored byA₂₈₀.

[0418] Multi-PEGylated species are separated by applying a lineargradient of 0-50% Buffer B for 180 minutes at a flow rate of 4 ml/minand collecting 2 ml fractions. The collected fraction are analyzed bySDS-PAGE, and fractions having a desired number of attached PEG groupsare pooled. This allows purification of a PEGylated G-CSF mixturecomprising species initially having, e.g., 3-6 attached PEG groups toresult in a product having e.g. only 4 or 5 PEG groups attached, or aproduct having only a single number of attached PEG groups.

EXAMPLE 19

[0419] Peptide Mapping

[0420] Using a similar procedure to that described above in Example 7,but based on degradation with trypsin, the PEGylation pattern of a G-CSFconjugate of the invention was determined by peptide mapping. In thiscase, the polypeptide was produced in CHO cells (see Example 3) and hadthe substitutions K16R, K34R, K40R, T 105K and S159K relative to thesequence of native human G-CSF. It was PEGylated with 5 kDa SPA-PEG asdescribed above, resulting in modified proteins carrying predominantly3, 4 or 5 PEG moieties, and to a small extent 6 PEG moieties. Five ofthe six possible PEG attachment sites are known, these being theN-terminal amino group, Lys23, Lys105, Lys159 and His170.

[0421] This peptide mapping analysis showed that the conjugated proteinwas essentially fully PEGylated at the N-terminal and at Lys105 andLys159, while Lys23 was partially PEGylated. Although His170 has beenshown to be partially PEGylated in previous experiments, this wassurprisingly not found in this experiment. One possible explanation forthis observation is that the bond between the PEG and the His170 residuemay be unstable during the sample preparation carried out prior to thepeptide mapping. A possible unstable PEGylation such as may be the casehere may be avoided by substituting the histidine residue with anotherresidue, in particular a lysine residue if a more stable PEGylation isdesired, or a glutamine or arginine residue if PEGylation is to beavoided.

EXAMPLE 20

[0422] In vivo Biological Activity in Rats with Chemotherapy-InducedNeutropenia

[0423] The in vivo biological activity of two PEGylated G-CSF variantsof the invention was tested in rats with chemotherapy-inducedneutropenia. The variants had, relative to SEQ ID NO:1, the amino acidsubstitutions K16R, K34R, K40R, T105K and S159K (referred to below as“105/159”) and K16R, K34R, K40R, Q90K, T105K and S159K (referred to as“90/105/159”), respectively. Both variants were produced in yeast (S.cerevisiae) and were conjugated with SPA-PEG-5000 as described above.The in vivo biological activity of a single dose of the two variants wastested against the activity of daily doses of non-conjugated hG-CSF(Neupogen®) and a control (vehicle).

[0424] 24 hours before administration of the G-CSF samples, the ratswere given 50 mg per kg body weight of CPA. The PEGylated variants ofthe invention were administered as a single dose of 100 μg per kg bodyweight at time 0, while Neupogen® was administered in daily doses of 30μg per kg body weight for 5 days (from 0 hours to 96 hours).

[0425] The in vivo biological activity was measured as described above(“Measurement of the in vivo biological activity in rats withchemotherapy-induced neutropenia of conjugated and non-conjugated hG-CSFand variants thereof”). The results are shown in FIG. 8 (white bloodcell count, WBC) and in FIG. 9 (absolute neutrophil count, ANC).

[0426] As seen in FIG. 8, administration of 105/159, 90/105/159 andNeupogen® all resulted in an initial increase in white blood cell levelsin the first 12 hours, after which the white blood cell levels fell as aresult of the chemotherapy, reaching a minimum after about 48 hours.After 48 hours, the numbers of white blood cells increased for all threetreatment groups, although the rate of increase was clearly greater forthe group treated with the two PEGylated variants of the invention thanfor the group treated with Neupogen®. Treatment with the PEGylatedvariants 105/159 and 90/105/159 resulted in a normal level of whiteblood cells (over 10×10⁹/l) after 96 hours, while the Neupogen® treatedgroup still had a white blood cell level under 10×10⁹/l after 120 hours.Since the last of the five daily Neupogen® treatments was given at 96hours, the white blood cell level in this group fell again after 120hours. In contrast, the white blood cell level in the two groups treatedwith a single dose of the PEGylated variants of the invention wasrelatively stable at just over 10×10⁹/l from 96 hours and for theduration of the experiment until 216 hours.

[0427] A similar pattern for the numbers of neutrophils is seen in FIG.9, which shows that the neutrophil level for the group treated with thePEGylated variant 105/159 increased significantly faster than for thegroup treated with Neupogen® (ANC was not determined for the 90/105/159group).

EXAMPLE 21

[0428] In vivo Biological Activity in Rats with Chemotherapy-InducedNeutropenia

[0429] The in vivo biological activities of non-conjugated hG-CSF(Neupogen®) and hG-CSF with a single N-terminally linked 20 kDa PEGgroup (Neulasta™) were compared to two PEGylated G-CSF variants of theinvention in rats with chemotherapy-induced neutropenia. These twovariants, which were produced in yeast (S. cerevisiae) and CHO cells,respectively, had the same amino acid substitutions relative to thesequence of hG-CSF, namely K16R, K34R, K40R, T105K and S159K, and wereconjugated to SPA-PEG 5000. The PEGylated variants of the invention,which initially consisted of multi-PEGylated species having 3-6 PEGmoieties attached, were separated to give a more uniform product havingonly 4-5 PEG moieties attached. These variants are referred to below as“G20” (produced in yeast) and “G21” (produced in CHO cells).

[0430] The G-CSF samples were administered 24 hours after administrationof CPA (90 mg per kg body weight). The PEGylated variants, i.e.Neulasta™, G20 and G21, were administered as a single dose of 100 μg perkg body weight, while Neupogen® was administered in daily doses of 101 gper kg body weight for seven days.

[0431] The in vivo biological activity was measured as described above(“Measurement of the in vivo biological activity in rats withchemotherapy-induced neutropenia of conjugated and non-conjugated hG-CSFand variants thereof”). The results are shown in FIG. 10 (white bloodcell count, WBC) and FIG. 11 (absolute neutrophil count, ANC).

[0432]FIGS. 10 and 11 show that all of the G-CSF compounds induced aninitial formation of white blood cells and neutrophils at approximatelyidentical rates during the first 12 hours, after which the levels ofwhite blood cells and neutrophils fell as a result of the chemotherapy.After 96 hours, the levels of white blood cells and neutrophilsincreased once again in all cases, but the rate of increase wassignificantly higher for rats treated with G20 or G21 than for ratstreated with either Neupogen® or Neulasta™. FIG. 10 shows that the whiteblood cell levels of rats treated with G20 or G21 reached a normal levelof approximately 109/1 after 144 hours, while the rats treated withNeupogen® or Neulastam did not reach this level until after 168 hours.As shown in FIG. 11, the same pattern is seen when looking at theneutrophil count, i.e. the neutrophil count of rats treated with G20 orG21 reach a normal level approximately 24 hours before rats treated withNeupogen®) or Neulasta™ reach a similar level. It may thus be concludedthat these PEGylated G-CSF variants of the invention are able to reducethe duration of chemotherapy-induced neutropenia in rats by about 24hours compared to treatment with the currently available G-CSF productsNeupogen® and Neulasta™.

[0433] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims. Forexample, all the techniques and apparatus described above may be used invarious combinations. All publications, patents, patent applications,and/or other documents cited in this application are incorporated hereinby reference in their entirety for all purposes to the same extent as ifeach individual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated herein byreference in its entirety for all purposes.

1 15 1 174 PRT Homo sapiens 1 Thr Pro Leu Gly Pro Ala Ser Ser Leu ProGln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile GlnGly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys Ala Thr Tyr Lys LeuCys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His Ser Leu Gly Ile ProTrp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser Gln Ala Leu Gln Leu Ala GlyCys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe Leu Tyr Gln Gly LeuLeu Gln Ala Leu Glu Gly Ile Ser 85 90 95 Pro Glu Leu Gly Pro Thr Leu AspThr Leu Gln Leu Asp Val Ala Asp 100 105 110 Phe Ala Thr Thr Ile Trp GlnGln Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Ala Leu Gln Pro Thr GlnGly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 Gln Arg Arg Ala GlyGly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155 160 Leu Glu ValSer Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 2 525 DNA ArtificialSequence misc_feature DNA sequence encoding hG-CSF, with codon usage forE. coli 2 acccctctgg gcccggccag cagtctgcct cagagttttt tactgaaatgcttagaacag 60 gtgcgtaaaa tccagggcga tggcgcggcc ctgcaggaaa aactgtgcgcgacctataaa 120 ctgtgccatc ctgaagaact ggtcctgtta ggccatagct taggcatcccgtgggcgcct 180 ctgagtagct gcccgagtca ggccctgcag ctggccggct gcctgagtcagttacatagt 240 ggcttatttt tatatcaggg cttactgcag gcgttagaag gcattagtccggaactgggc 300 ccgaccctgg ataccttaca gttagatgtc gcggattttg ccaccaccatttggcagcag 360 atggaagaat taggcatggc gcctgcgtta cagcctaccc agggcgccatgcctgcgttt 420 gcgagtgcgt ttcagcgtcg cgccggcggc gtgttagtgg ccagccatctgcagagcttt 480 ctggaagtga gttatcgtgt gttacgccat ctggcccagc cttaa 525 321 PRT Escherichia coli 3 Met Lys Lys Thr Ala Ile Ala Ile Ala Val AlaLeu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala 20 4 63 DNA ArtificialSequence misc_feature DNA sequence encoding the OmpA signal sequence 4atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60 gcc63 5 15 PRT Artificial Sequence PEPTIDE (1)..(15) Synthetic tag 5 MetLys His Gln His Gln His Gln His Gln His Gln His Gln Gln 1 5 10 15 6 45DNA Artificial Sequence misc_feature DNA encoding the tag of SEQ ID NO56 atgaaacacc aacaccaaca tcaacatcaa catcaacatc aacag 45 7 30 PRT Homosapiens SIGNAL (1)..(30) 7 Met Ala Gly Pro Ala Thr Gln Ser Pro Met LysLeu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp ThrVal Gln Glu Ala 20 25 30 8 615 DNA Artificial Sequence misc_featureSequence encoding hG-CSF with signal peptide of SEQ ID NO7 8 atggccggccctgccacaca gtcccccatg aagctgatgg ccctgcagct gctgctgtgg 60 cactccgccctgtggacagt gcaggaggcc acccctctgg gccccgccag ctccctgcct 120 cagtccttcctgctgaagtg cctggagcag gtgagaaaga tccagggcga cggcgccgcc 180 ctgcaggagaagctgtgcgc cacatacaag ctgtgccacc ctgaggagct ggtgctgctg 240 ggccacagcctgggcatccc ctgggcccct ctgtccagct gcccctccca ggccctgcag 300 ctggccggctgcctgtccca gctgcactcc ggcctgttcc tgtaccaggg cctgctgcag 360 gccctggagggcatctcccc cgagctgggc cccacactgg ataccctgca gctggacgtg 420 gccgatttcgccaccacaat ctggcagcag atggaggagc tgggcatggc ccctgccctg 480 cagcctacccagggcgccat gcctgccttt gcctccgcct ttcagagacg ggccggcggc 540 gtgctggtggccagccacct gcagagcttt ctggaggtgt cctacagagt gctgcggcac 600 ctggcccagccttga 615 9 6 PRT Artificial Sequence PEPTIDE (1)..(6) Synthetic tag 9His His His His His His 1 5 10 8 PRT Artificial Sequence PEPTIDE(1)..(8) Synthetic tag 10 Met Lys His His His His His His 1 5 11 10 PRTArtificial Sequence PEPTIDE (1)..(10) Synthetic tag 11 Met Lys His HisAla His His Gln His His 1 5 10 12 14 PRT Artificial Sequence PEPTIDE(1)..(14) Synthetic tag 12 Met Lys His Gln His Gln His Gln His Gln HisGln His Gln 1 5 10 13 10 PRT Artificial Sequence PEPTIDE (1)..(10)Synthetic tag 13 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 14 8 PRTArtificial Sequence PEPTIDE (1)..(8) Synthetic tag 14 Asp Tyr Lys AspAsp Asp Asp Lys 1 5 15 9 PRT Artificial Sequence PEPTIDE (1)..(9)Synthetic tag 15 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5

We claim:
 1. A polypeptide conjugate exhibiting G-CSF activity,comprising a polypeptide comprising at least one substitution selectedfrom the group consisting of K16R/Q, K34R/Q and K40R/Q, and at least onesubstitution selected from the group consisting of T105K and S159K,relative to the amino acid sequence of hG-CSF shown in SEQ ID NO: 1 orin a corresponding position relative to an amino acid sequence having atleast 80% sequence identity with SEQ ID NO: 1, the conjugate having 2-6polyethylene glycol moieties with a molecular weight of about1000-10,000 Da attached to attachment groups of the polypeptide.
 2. Thepolypeptide conjugate of claim 1, comprising three, four or five of saidsubstitutions.
 3. The polypeptide conjugate of claim 1, furthercomprising the substitution H170K, H170Q or H170R.
 4. The polypeptideconjugate of claim 1, comprising substitutions selected from the groupconsisting of: Q70K+S159K, Q70K+H170K, Q90K+S 159K, Q90K+H170K,T105K+S159K, T105K+H170K, Q120K+S159K, Q120K+H170K, T133K+S159K,T133K+H170K, S159K+H170K, Q70K+Q90K+S159K, Q70K+Q90K+H170K,Q70K+T105K+S159K, Q70K+T105K+H170K, Q70K+Q120K+S159K, Q70K+Q120K+H170K,Q70K+T133K+S159K, Q70K+T133K+H170K, Q70K+S159K+H170K, Q90K+T105K+S159K,Q90K+T105K+H170K, Q90K+Q120K+S 159K, Q90K+Q120K+H170K, Q90K+T133K+S159K, Q90K+T133K+H170K, Q90+S159K+H170K, T105K+Q120K+S159K,T105K+Q120K+H170K, T105K+T133K+S159K, T105K+T133K+H170K,T105K+S159K+H170K, Q120K+T133K+S159K, Q120K+T133K+H170K,Q120K+S159K+H170K, T133K+S159K+H170K, Q70K+Q90K+T105K+S159K,Q70K+Q90K+T105K+H170K, Q70K+Q90K+Q120K+S159K, Q70K+Q90K+Q120K+H170K,Q70K+Q90K+T133K+S159K, Q70K+Q90K+T133K+H170K, Q70K+Q90K+S159K+H170K,Q70K+T105K+Q120K+S159K, Q70K+T105K+Q120K+H170K, Q70K+T105K+T133K+S159K,Q70K+T105K+T133K+H170K, Q70K+T105K+S159K+H170K, Q70K+Q120K+T133K+S159K,Q70K+Q120K+T133K+H170K, Q70K+T133K+S159K+H170K, Q90K+T105K+Q120K+S159K,Q90K+T105K+Q120K+H170K, Q90K+T105+T133K+S159K, Q90K+T105+T133K+H170K,Q90K+T105+S159K+H170K, Q90K+Q120K+T133K+S159K, Q90K+Q120K+T133K+H170K,Q90K+Q120K+S159K+H170K, Q90K+T133K+S159K+H170K, T105K+Q120K+T133K+S159K,T105K+Q120K+T133K+H170K, T105K+Q120K+S159K+H170K,T105K+T133K+S159K+H170K and Q120K+T133K+S159K+H170K.
 5. The polypeptideconjugate of claim 1, comprising substitutions selected from the groupconsisting of K16R+K23R, K16R+K34R, K16R+K40R, K23R+K34R, K23R+K40R,K34R+K40R, K16R+K23R+K34R, K16R+K23R+K40R, K23R+K34R+K40R,K16R+K34R+K40R and K16R+K23R+K34R+K40R.
 6. The polypeptide conjugate ofclaim 5, comprising the substitutions K16R+K34R+K40R, and comprising alysine residue in position
 23. 7. The polypeptide conjugate of claim 1,wherein said substitutions are in a corresponding position relative toan amino acid sequence having at least about 90% sequence identity withSEQ ID NO:1.
 8. The polypeptide conjugate of claim 7, wherein saidsubstitutions are in a corresponding position relative to an amino acidsequence having at least about 95% sequence identity with SEQ ID NO:
 19. The polypeptide conjugate of claim 1, wherein the polyethylene glycolmoieties are attached to at least one lysine residue and optionally alsoto the N-terminal amino group.
 10. The polypeptide conjugate of claim 1,comprising a polypeptide comprising the substitutions K16R, K34R, K40R,T105K and S159K relative to the amino acid sequence of hG-CSF shown inSEQ ID NO:1 and having 2-6 polyethylene glycol moieties with a molecularweight of about 1000-10,000 Da attached to attachment groups of thepolypeptide.
 11. The polypeptide conjugate of claim 1, which isglycosylated at T133.
 12. The polypeptide conjugate of claim 1, having3-5,4-6, 3-4,4-5 or 5-6 attached PEG moieties.
 13. The polypeptideconjugate of claim 12, having 3-6 polyethylene glycol moieties with amolecular weight of about 5000-6000 Da attached.
 14. The polypeptideconjugate of claim 13, having 4 PEG moieties of about 5 kDa attached.15. The polypeptide conjugate of claim 13, having 5 PEG moieties ofabout 5 kDa attached.
 16. The polypeptide conjugate of claim 1, havingan in vitro bioactivity in the range of about 2-30% of the bioactivityof non-conjugated hG-CSF as determined by the luciferase assay describedherein.
 17. A method for preparing a G-CSF conjugate which provides areduced duration of neutropenia compared to hG-CSF, the methodcomprising preparing a polypeptide comprising an amino acid sequencethat differs in at least one amino acid residue from the amino acidsequence of hG-CSF shown in SEQ ID NO: 1, and attaching anon-polypeptide moiety to each of at least two attachment groups of saidpolypeptide to result in a conjugate having an in vitro bioactivity inthe range of about 2-30% of the bioactivity of non-conjugated hG-CSF asdetermined by the luciferase assay described herein.
 18. The method ofclaim 17, wherein a desired in vitro bioactivity is obtained byalteration of at least one amino acid residue selected from amino acidposition 11-41 (helix A), 71-95 (helix B), 102-125 (helix C), and145-170 (helix D), relative to the amino acid sequence of SEQ ID NO: 1,and by conjugation of at least one non-polypeptide moiety to the atleast one altered amino acid residue.
 19. A composition comprising thepolypeptide conjugate of claim 1 and at least one pharmaceuticallyacceptable carrier or excipient.
 20. A method for treating a mammalsuffering from an insufficient neutrophil level, comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of the polypeptide conjugate of claim 1.